CN103333819A - Bacillus subtilis capable of producing broad spectrum and efficient antibacterial peptide and application of Bacillus subtilis - Google Patents
Bacillus subtilis capable of producing broad spectrum and efficient antibacterial peptide and application of Bacillus subtilis Download PDFInfo
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Abstract
The invention discloses Bacillus subtilis capable of producing broad spectrum and efficient antibacterial peptide and an application of the Bacillus subtilis and belongs to the technical field of microorganisms, wherein the Bacillus subtilis is Bacillus subtilis BRT39 concretely with the preservation number of CGMCC NO. 5702. Antibacterial peptide in metabolic products of a BRT39 bacterial strain has stronger anti-bacterial effect, a fermentation liquor can inhibit gram positive bacteria and also has stronger inhibiting effect on multiple gram negative bacteria and fungi, and the antibacterial peptide in the fermentation liquor has strong acid-base stability, good thermal stability, strong light stability and high organic solvent tolerance. The antibacterial peptide produced by the BRT39 bacterial strain belongs to a natural antibacterial agent, has no side or toxic effect, can be used as a food preservative, a feed additive, an agricultural biological control agent and an animal disease prevention agent and has the advantages of no toxicity, no residue, no drug resistance and high safety.
Description
Technical field
The invention belongs to microbial technology field, specifically is subtilis and application thereof that a strain produces the broad-spectrum high efficacy antibacterial peptide.
Background technology
Under the suitable situation of envrionment conditions, the microorganism in the food breeds in a large number, causes food spoilage.One of key issue of food-processing is anti-corrosive fresh-keeping.And wanting to reach the prolongation preservation term, one of important measures that make the food circulation and have commodity value are to add an amount of food preservative freshness retaining agent in food processing process.Chemical Preservative such as phenylformic acid, Sorbic Acid and its esters, parabens etc. are to use food preservatives the most widely, can play and suppress or killing microorganisms, stop, delay the putrid and deteriorated of food.Different with industrial sanitas, the safety and sanitation condition of food preservatives is subjected to people day by day and pays close attention to, and the human consumer feels scruple to the food that adds Chemical Preservative.Therefore, seeking wide spectrum, efficient, low toxicity, whole food antisepsis antistaling agent is one of focus in the present food scientific research.The potential potential safety hazard of Chemical Preservative can not be ignored, and seeks a kind of new surrogate and is attracted attention by various countries' researcher to reduce the potential hazard that Chemical Preservative was caused.Therefore, efficient, safe, the nontoxic food preservatives of research and development becomes the developing direction of food preservatives.Antiseptics for natural food is as class new type of safe sanitas efficiently, become one of focus of food scientific research, antibacterial peptide domestic and international research work at present mainly concentrates on the application of industry aspects such as medicine, agricultural, livestock industry, and the research aspect food antiseptic is very few; And antibacterial peptide is many extracts from materials such as plant, insect, because of extract the limited and extraction process complexity of raw material, cost is higher, is difficult to form industrialization.By microbial fermentation, obtaining highly active antibacterial peptide is the production technology with important prospect.At present, existing many natural antibacterial peptides are studied and find, but the more bacteriocin of applied research has only Buddhist nun's suffering (Nisin) and tennecetin (Natamycin) in food, and the applied research of other antibacterial peptides is less.
Summary of the invention
The purpose of this invention is to provide subtilis and application thereof that a strain produces the broad-spectrum high efficacy antibacterial peptide.
Bacterial strain with anti-microbial effect provided by the invention is specially subtilis (Bacillus subtilis) BRT39, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 9th, 2012 and (is called for short CGMCC, the address is: No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city, postcode: 100101), preserving number is CGMCCNO.5702.
Described BRT39 bacterial strain is 37 ℃ of cultivation 24h on the LB solid medium, and bacterium colony is rounded, dirty white, and the edge is irregular, and is opaque, and surface folding is rough, the irregular expansion in edge.Peritrichous can move, and no pod membrane has gemma, and the thalline size is (0.7～0.9) μ m * (3～3.5) μ m, gemma (0.6～0.9) μ m * (1.0～1.5) μ m, and ellipticity is positioned at thalline central authorities or inclined to one side slightly, and gemma forms the back thalline and does not expand.When growing in the liquid nutrient medium, form the wrinkle mould.Casein hydrolysis experiment, starch hydrolysising experiment, gelatine liquefication experiment, nitrate reduction are tested all positive; Thalline can utilize glucose, fructose, sucrose, can not utilize lactose and N.F,USP MANNITOL; H
2S, catalase experiment, indole test are tested all negative.
Described BRT39 bacterial strain 16sRNA gene checks order.Resulting sequence is carried out homology relatively with existing 16SrDNA sequence among Blast software and the Genbank, utilize software MEGA4.0 to make up evolutionary tree, the result shows that it is one that bacterial strain of the present invention and subtilis Bacillus Subtilis (GQ36007543578) gather, sequence similarity is 98%, the results are shown in Figure 1.Morphological feature, physiological and biochemical property and the 16S rDNA nucleotide sequence homology analytical results of comprehensive BRT39 bacterial strain are accredited as subtilis (Bacillus Subtilis) with bacterial strain BRT39.
Antibacterial peptide has strong bacteriostatic action in described its meta-bolites of BRT39 bacterial strain, its fermented liquid not only can suppress gram positive bacterium, simultaneously multiple gram negative bacterium and fungi is all had stronger restraining effect: to streptococcus aureus (Staphylococcus aureaus), intestinal bacteria (Escherichia coli), bacillus cereus (Bacillus cereus), Salmonellas (Salmonella enteritidis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), subtilis bacteriums such as (Bacillus subtilis) has showed stronger restraining effect; Aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), viride (Trichoderma viride), Mucor racemosus filamentous funguss such as (Mucor racemosus) there is strong restraining effect.
The antibacterial peptide ph stability is strong in described its meta-bolites of BRT39 bacterial strain, and under the environment of pH212, behind the effect 1h, anti-microbial activity is almost constant; The antibacterial peptide light stability is strong, uviolizing 5h, and anti-microbial activity is constant; Better heat stability is handled 1h for 80 ℃, and bacteriostatic activity does not change; After organic solvent such as acetone, chloroform, ethyl acetate, methyl alcohol, ether were handled 30min, the antibacterial substance activity remained unchanged.Antibacterial peptide is responsive to proteolytic enzyme, uses respectively in papoid, plant protease, aspartic protease, Sumizyme MP, the pepsin meta-bolites behind the antibacterial peptide, and bacteriostatic activity descends.
Beneficial effect
1, bacterial strain BRT39 of the present invention belongs in the world the food safety bacterial classification of generally acknowledging, is 42 kinds of U.S. FDA issue in 1989 one of probioticses safely and effectively, is simultaneously to allow one of 12 kinds of probiotic bacteriums of using in the food announced in 1996 of China Ministry of Agriculture.Antibacterial peptide that bacterial strain of the present invention produces belongs to natural antibacterial agent, has no side effect, and can be used as food preservatives, fodder additives, agricultural biological control agent and animal diseases prevention agent, has nontoxic, noresidue, does not produce chemical sproof advantage.Meet current green safety foodstuff production and environment amenable requirement.Therefore, has high security in actual use.
2, bacterial strain BRT39 of the present invention produces the antibacterial peptide has a broad antifungal spectrum, and bacteriums such as common pathogenic bacteria in food such as streptococcus aureus, intestinal bacteria, bacillus cereus, Salmonellas, Pseudomonas aeruginosa have been showed stronger restraining effect; Also can effectively suppress filamentous funguss such as aspergillus niger, aspergillus oryzae, viride, Mucor racemosus.So can be in food Secure Application.
3, to produce antiseptic peptide stability strong for bacterial strain BRT39 of the present invention.The existing antibacterial peptide that uses of its scope of application is wide.Under the environment of pH2-12, behind the effect 1h, anti-microbial activity is almost constant; Heating 30min bacteriostatic activity does not change under 100 ℃ of conditions, has shown in food and feed high temperature process process the superiority that bacteriostatic activity is not lost.After organic solvent such as acetone, chloroform, ethyl acetate, methyl alcohol, ether were handled 30min, the antibacterial substance activity remained unchanged.
4, the present invention adopts subtilis to produce antibacterial peptide, not limited by raw material, and is with short production cycle, cost is low, but large-scale industrial production, and be not subjected to the influence of external environments such as season and climate change has clear superiority with respect to the antibacterial peptide of plant and animal material.
Description of drawings
Fig. 1 bacterial strain BRT39 phylogenetic evolution tree
Fig. 2 bacterial strain BRT39 produces antibacterial peptide milk corrosion-resistanting test design sketch
Embodiment:
Below by specific embodiment narration the present invention.Unless stated otherwise, used technique means is method known in those skilled in the art among the present invention.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by claims.To those skilled in the art, under the prerequisite that does not deviate from essence of the present invention and scope, various changes that the material component in these embodiments and consumption are carried out or change and also belong to protection scope of the present invention.
The screening of embodiment 1:BRT39 bacterial strain
(1) gets the 2g soil sample from the abundant vegetable garden of organic content, be added in the little triangular flask that contains 50mL cooling sterilized water, shaking table 200r/min, shake 20min, be placed on then in 80 ℃ of water-baths, water-bath 10min constantly shakes triangular flask mixing soil sample, leave standstill 5min and draw supernatant liquor 100 μ L, gradient dilutions to 10 successively
-1～10
-9Concentration chooses 10
-3, 10
-4, 10
-5, 10
-6Concentration coating beef extract-peptone flat board is cultivated 24h for 37 ℃, continues 30 ℃ and cultivates 24h.
(2) but according to the feature of the aspects such as lysochrome of shape, size, surface tissue, marginal texture, quality, gloss, transparency, color and the generation of microbe colony, choose with the single bacterium colony that transfering loop will isolate, move on on the screening culture medium flat board, and numbering, 48h cultivated for 34 ℃.
Described screening culture medium consists of: extractum carnis 3g, sodium-chlor 5g, peptone 10g, agar powder 15g, distilled water 1000mL, pH7.2,121 ℃ of sterilization 20min.
(3) treat that bacterial strain after flat board is cultivated 48h, covers the adding minimum of chloroform at culture dish, the heavily smoked 50min of left-hand thread, fully blow away remaining chloroform after, add the indicator liquid of 2～3 times of dilutions, culture dish is half-open, does the back until planar surface and cultivates 16h in 37 ℃.After selecting the bigger single bacterium colony of bacterial strain line separation of inhibition zone, preservation is used for multiple sieve.
(4) after the bacterial strain of preservation is received slant activation 2 times, the good bacterial classification of activation is inserted seed culture medium, 180r/min, cultivates 18h by 37 ℃.Be inoculated into fermention medium by 5% inoculum size again, 200r/min, 28 ℃, fermentation 48h.With the centrifugal thalline that goes of gained fermented liquid 10000r/min, fermented liquid supernatant liquid carries out bacteriostatic experiment.
Described seed fermentation substratum consists of: glucose 2g, peptone 1g, NaH
2PO
42H
2O0.2g, Na
2HPO
42H
2O0.4g, MgSO
47H
2O0.05g, distilled water 100ml, pH7.4,121 ℃ of sterilization 20min.
Described fermention medium consists of: glucose 2g, peptone 2g, NaH
2PO
42H
2O0.02g, Na
2HPO
42H
2O0.04g, MgSO
47H
2O0.01g, CaCl
20.01g, MnSO
40.002g, distilled water 100mL pH7.4,121 ℃ of sterilization 20min.
(5) add indicator nutrient solution (OD600=1.6) at the beef extract-peptone flat board, dry the back and punch with aperture 6.0mm punch tool, every hole adds the about 100 μ L of different strain fermented liquid supernatant liquid respectively, cultivates 16h for 37 ℃, measures the inhibition zone size.When detecting the mould bacteriostatic activity, mould indicator on PDA culture medium flat plate central point, 28 ℃ are cultured to about colony diameter 2cm, and the punching of 0.5cm place adds fermented liquid supernatant liquid around it, and the inhibition zone size is measured in 28 ℃ of cultivations.
(6) select that bacteriostatic activity is strong, the bacterial strain of antibiotic wide spectrum carries out glycerine and preserves.
Embodiment 2: identification of strains
The bacterial strain Physiology and biochemistry is identified: according to " uncle's Jie Shi Bacteria Identification handbook (the 9th edition) method is carried out tests such as utilization of carbon source test, catalase test, hydrogen sulfide production test, indole test, nitrate and nitrite reduction test; Morphology is identified: dibbling is on the beef-protein medium solid plate respectively with bacterial strain to be identified, and 37 ℃ of cultivation 2d describe also record bacterium colony cultural characteristic and morphological features.The Physiology and biochemistry identification mark is as shown in table 1." uncle Jie Shi Bacteria Identification handbook conforms to subtilis kind feature in result's contrast in the table.The extraction of molecular biology identification: bacterial genomes DNA is with reference to the method for " molecular cloning experiment guide " (the 3rd edition).According to sequences Design primer the most conservative among the bacterial 16 S rDNA.Primers F: 5 '-AGAGTT TGA TCM TGGCTC AG-3 '; Primer R:5 '-AAG GAG GTG WTCCAR CC-3 ' gives birth to worker's biotechnology company limited by Shanghai and synthesizes.The PCR reaction conditions: 94 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 40s, 72 ℃ of 2min, 30 circulations, 72 ℃ are extended 10min eventually.Pcr amplification product is delivered to the order-checking of Beijing three rich polygala root Bioisystech Co., Ltd.After sequencing result carries out two-way splicing, the 16S rDNA sequence that obtains is submitted to carries out the BLAST on-line analysis in the NCBI nucleic acid database.The part genus bacillus 16S rDNA sequence in the picked at random report from the GenBank nucleic acid database of NCBI is used Clustal X1.81 and Phylodraw082 software building systematic evolution tree and analysis afterwards, and phylogenetic tree as shown in Figure 1.In conjunction with form and the physiological and biochemical property of bacterial strain, preliminary evaluation is fixed tentatively the BRT39 bacterial strain and is subtilis.
Table 1 bacterial strain BRT39 morphology and physio-biochemical characteristics
Annotate: result "+" represents positive; "-" expression is negative.
Embodiment 3: the determination of activity of bacterial strain fermentation liquor antimicrobial spectrum
(1) fermentation culture of bacterial strain BRT39 through the centrifugal 5min of 10000r/min, obtains supernatant liquor behind 0.22 μ m filtering with microporous membrane, is used for anti-microbial activity and measures.
(2) adopt nutrient agar to cultivate indicator, directly measure shake flask fermentation liquid anti-microbial activity with punch method.The cultured 100 μ L indicator bacterium suspension (1 * 10 of the dull and stereotyped coating of nutrient agar
8Cfu/ml), evenly punch with diameter 6mm punch tool, every hole adds the fermented liquid 100 μ L that prepare, and does contrast with sterilized water.Cultivate 16h for 37 ℃, record inhibition zone size.Select intestinal bacteria (Escherichia coli), golden yellow grape ball (Staphylococcus aureaus), bacillus cereus (Bacillus cereus), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Salmonellas (Salmonella enteritidis), subtilis bacteriums such as (Bacillus subtilis) as strain subject.When doing the mould bacteriostatic experiment, inoculation mould in the middle of the PDA culture medium flat plate behind the cultivation 1-2d, is being punched from bacterium colony 2cm place, and every hole adds the fermented liquid 100 μ L that prepare, and does contrast with sterilized water.Cultivate 3-4d, record antibacterial situation for 28 ℃.
(3) the antibacterial substance bacteriostatic activity sees Table 2 in the bacterial strain BRT39 fermentation culture, show fermented liquid to and intestinal bacteria (Escherichia coli), streptococcus aureus (Staphylococcus aureaus), bacillus cereus (Bacillus cereus), Salmonellas (Salmonella enteritidis) and subtilis prokaryotic micro-organisms such as (Pseudomonas aeruginosa) had the good restraining effect.Filamentous fungus aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), viride (Trichoderma viride), Mucor racemosus (Mucor racemosus) also had significant inhibitory effect.
Table 2 bacterial strain BRT39 fermentation culture bacteriostatic activity result
| Suppress bacterial strain | Antibacterial circle diameter (mm) |
| Intestinal bacteria (Escherichia coli) | 21 |
| Streptococcus aureus (Staphylococcus aureaus) | 24 |
| Bacillus cereus (Bacillus cereus) | 20 |
| Salmonellas (Salmonella enteritidis) | 23 |
| Pseudomonas aeruginosa (Pseudomonas aeruginosa) | 16 |
| Subtilis (Bacillus subtilis) | 20 |
| Aspergillus niger (Aspergillus niger) | 17 |
| Aspergillus oryzae (Aspergillus oryzae) | 18 |
| Viride (Trichoderma viride) | 17 |
| Mucor racemosus (Mucor racemosus) | 21 |
Embodiment 4: the determining of antibacterial peptide
Adopt internationally recognized " the responsive method of proteolytic enzyme " to determine that antimicrobial substance is antibacterial peptide.The centrifugal 10min of fermented liquid 12000r/min with embodiment 3 obtains keeps supernatant liquor, and removes thalline by 0.22 μ m membrane filtration.Fermented supernatant fluid pH value is adjusted to about 7.5, gets rid of bacterial strain and produce organic acid or alkali during the fermentation to the influence of fungistatic effect.Proteinase K, stomach en-, proteolytic enzyme E, papoid, trypsinase are made into the solution of 10mg/ml respectively, get 1mL respectively in centrifuge tube, add the fermented supernatant fluid 1ml that handles well again, the final concentration that makes enzyme is 5mg/mL, simultaneously add the 1mL fermented liquid in contrast with the 1mL distilled water, 37 ℃ of incubations 4 hours are hatched and are finished to boil the enzyme 5min that goes out in back 100 ℃ of boiling water baths, then measure antibacterial circle diameter with agar diffusion method, and compare with blank.Observe the variation of bacteriostatic activity.The result is as shown in table 3.This antibacterial substance is very inresponsive to proteolytic enzyme E, papoid, but responsive especially to stomach en-, Proteinase K, trypsinase, activity is almost completely lost.By above test-results analysis-by-synthesis, judge that this antibacterial substance is antibacterial peptide.
The different protease treatment of table 3 are to the influence of antimicrobial substance
Embodiment 5: the bacteriostatic activity of bacterial strain fermentation liquor different treatment
(1) bacterial strain BRT39 fermented liquid antibacterial peptide thermal stability determination is in 40 ℃, and 60 ℃, 80 ℃, under 100 ℃ of conditions the antibacterial peptide fermented liquid is handled 1h respectively, be incubated 20min under 120 ℃ of conditions.Be indicator strain with the streptococcus aureus, measure bacteriostatic activity by dull and stereotyped punch method, every hole adds antibacterial peptide fermented liquid 60 μ l, to do blank without heat treated antibacterial peptide fermented liquid.The result shows, when the antibacterial peptide fermented liquid is handled 1h down through 40 ℃, 60 ℃, 80 ℃, 100 ℃, antibacterial peptide fermented liquid bacteriostatic activity is with showing that without heat treated contrast bacteriostatic activity remains on more than 95%, and behind 120 ℃ of effect 20min, bacteriostatic activity drops to 7080%.This shows that the antibacterial peptide fermented liquid has stronger thermostability.
(2) bacterial strain BRT39 fermented liquid antibacterial peptide ph stability is measured the antibacterial peptide fermented liquid is transferred to 2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0,12.0,13.0 with soda acid respectively with pH, handle 16h for 4 ℃ then, with soda acid the antibacterial peptide fermented liquid is recalled to pH7.0 respectively again, be indicator strain with the streptococcus aureus, dull and stereotyped punch method detects antibacterial peptide fermented liquid bacteriostatic activity, and every hole adds antibacterial peptide fermented liquid 60 μ l.The result shows that between the pH4.0-9.0, bacteriostatic activity almost remains unchanged, and between pH2.0-4.0, bacteriostatic activity is reduced to 50-80%, and the pH10.0-13.0 bacteriostatic activity remains on 60-90%.So bacterial strain BRT39 fermented liquid antibacterial peptide ph stability a wider range.
(3) bacterial strain BRT39 fermented liquid antibacterial peptide organic solvent stability is measured the antibacterial peptide fermented liquid is mixed with isopyknic acetone, chloroform, ethanol, formaldehyde, ethyl acetate, ether, fully shake up effect 40min, in 55 ℃ of water-baths, remove organic solvent, detect the antibacterial peptide bacteriostatic action after respectively handling then.Be indicator strain with the streptococcus aureus, dull and stereotyped punch method detects antibacterial peptide fermented liquid bacteriostatic activity, and every hole adds antibacterial peptide fermented liquid 60 μ l.The result shows that the sample after acetone, chloroform, ethyl acetate, methyl alcohol, ether are handled does not have significant difference with the bacteriostatic activity of the sample that does not have to handle, and this shows that the fermented liquid antibacterial peptide is insensitive to these several organic solvents.
(4) bacterial strain BRT39 fermented liquid antibacterial peptide UV stable is measured the antibacterial peptide fermented liquid is placed under the 28W ultraviolet lamp, shine 1h, 3h, 5h, 8h, 10h, 12h respectively apart from the 10cm place, compare with the antibacterial peptide fermented liquid that does not carry out radiation treatment, detect it to the bacteriostatic activity of streptococcus aureus.Be indicator strain with the streptococcus aureus, dull and stereotyped punch method detects antibacterial peptide fermented liquid bacteriostatic activity, and every hole adds antibacterial peptide fermented liquid 60 μ l.The result shows that the antibacterial peptide fermented liquid that different time shone illustrates that with undosed anti-microbial activity no significant difference bacterial strain BRT39 fermented liquid antibacterial peptide UV stable is strong.
(5) bacterial strain BRT39 fermented liquid antimicrobial peptide protein enzyme stability is got the antibacterial peptide fermented liquid and is mixed with papoid, Sumizyme MP, trypsinase, stomach en-, plant protease, aspartic protease etc. respectively, the final concentration of proteolytic enzyme is 10 μ g/ml, enzymolysis 4h under the suitableeest action condition of enzyme separately is contrast with undressed antibacterial peptide fermented liquid.Be indicator strain with the streptococcus aureus, dull and stereotyped punch method detects antibacterial antiplaque peptide bacteriostatic activity, and every hole adds antibacterial peptide fermented liquid 60 μ l.The result shows that there were significant differences for the sample anti-microbial activity of processing and untreated sample anti-microbial activity, and other proteolytic enzyme has restraining effect to it except trypsinase, so antibacterial peptide is relatively more responsive to proteolytic enzyme.Preliminary definite antibacterial peptide is antibacterial protein.
Embodiment 6: the experiment of antibacterial peptide food antiseptic
With the fermented liquid that embodiment 3 obtains, adopt technologies such as ultrafiltration and concentration, cryodrying (＜100 ℃), micronizing to make the antibacterial peptide pressed powder, moisture＜5% is used for the food antiseptic test.
1, the anticorrosion experiment of milk
Ultra High Temperature Short Time milk with the supermarket purchase, join and clean up but in the unpasteurized test tube, every pipe 15mL, test tube is compiled 1～No. 7, the mass concentration that adds antibacterial peptide pressed powder 0.2g, 0.4g, 0.8g, 1.2g, 1.6g, 2.0g, 2.4g according to every 100ml milk in No. 1～7, experimental group joins in the above test tube, mixes.Blank does not add antibacterial peptide.Place 30d under the room temperature then, the putrid and deteriorated situation of record milk.The result as shown in Figure 2, behind the 30d, the invisible spectro milk of blank group is obviously rotten, color and luster is dark yellow, the water and milk layering produces flocks, opens the test tube plug, also smells one peculiar smell.1, corruption to a certain degree also appears in No. 2 test tubes, and the test tube of back is along with the increase of antibacterial peptide addition, and corruption is more and more not obvious, and wherein No. 7 test tube milk colors are pure white, free from extraneous odour, and sense organ is good, does not almost have denaturalization phenomenon.So this antibacterial peptide can be used for the anticorrosion of milk.
2, the anticorrosion experiment of strawberry juice
The mass concentration that adds antibacterial peptide pressed powder 0.2g, 0.4g, 0.8g, 1.2g, 1.6g, 2.0g, 2.4g according to every 100ml fresh strawberry juice joins in the fresh strawberry juice, mixes.Blank does not add antibacterial peptide.Place 10d under the room temperature then, observe strawberry juice sense organ and microbe number situation.The result is as shown in table 4, and along with the addition increase of antibacterial peptide, it is more and more lighter that the corruption of muddy and flocks appears in strawberry juice, is the above room temperature 10d of 1.2 (g/100ml) adding mass concentration, and the strawberry juice outward appearance does not have corruption, can accept.From content of microorganisms, add the breeding growth that antibacterial peptide can significantly suppress assorted bacterium, along with the interpolation of antibacterial peptide, microbe population is fewer and feweri.When antibacterial peptide adds mass concentration when 1.6 (g/100ml) are above, detect less than microorganism in the culture plate, when 1.6 (g/100ml) are above, can reach the fungistatic effect that suppresses microorganism growth fully so add mass concentration.
Table 4 subtilis BRT39 produces antibacterial peptide to the preservative effect of strawberry juice
Claims (2)
1. a strain produces the Bacillus subtilis strain of broad-spectrum high efficacy antibacterial peptide, and described bacterial strain is specially subtilis (Bacillus subtilis) BRT39, and preserving number is CGMCCNO.5702.
2. a strain as claimed in claim 1 produces the application of Bacillus subtilis strain CGMCC NO.5702 in food antiseptic of broad-spectrum high efficacy antibacterial peptide.
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| CN104109643A (en) * | 2014-04-23 | 2014-10-22 | 中国海洋大学 | Bacillus spp with broad-spectrum antibacterial activity |
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