CN101955902B - New brevibacillus brevis strain and application thereof - Google Patents
New brevibacillus brevis strain and application thereof Download PDFInfo
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- 235000000235 Euphoria longan Nutrition 0.000 claims abstract description 34
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 10
- 230000004151 fermentation Effects 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims description 26
- 241000233866 Fungi Species 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 23
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 20
- 230000001580 bacterial effect Effects 0.000 claims description 14
- 239000000843 powder Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000001888 Peptone Substances 0.000 claims description 9
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- 235000019319 peptone Nutrition 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 229920002472 Starch Polymers 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 235000015278 beef Nutrition 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
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- 235000019698 starch Nutrition 0.000 claims description 6
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- 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 claims description 3
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- 230000001954 sterilising effect Effects 0.000 claims description 2
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- 235000013399 edible fruits Nutrition 0.000 abstract description 18
- 230000003042 antagnostic effect Effects 0.000 abstract description 7
- 241000894006 Bacteria Species 0.000 abstract description 5
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- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 abstract description 4
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- 241000399352 Brevibacillus brevis FJAT-0809-GLX Species 0.000 abstract 4
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Abstract
The invention discloses a strain which has an antagonistic effect is obtained by separating and screening from watermelon rhizosphere soil in Yongtai County, Fujian Province. The strain is brevibacillus brevis strain FJAT-0809-GLX (Brevibacillus brevis). A microbial fresh-keeping bacterium agent which can keep longan fresh is obtained by adopting the fermentation liquor of the brevibacillus brevis strain FJAT-0809-GLX. The strain has the advantages that: a new brevibacillus brevis strain FJAT-0809-GLX is provided; the microbial fresh-keeping bacterium agent which can keep the longan fresh is prepared by the adopting the brevibacillus brevis strain FJAT-0809-GLX; and the bacterium agent can reduce the rotting rate of the longan and can keep the color, the luster and the taste of fruits.
Description
[technical field]
The present invention relates to a kind of mikrobe and application thereof, relate in particular to a kind of new short Bacillus strain and use its preparation longan mikrobe preserving fungus agent.
[background technology]
China is less through the fruits and vegetables ratio of fresh-retaining preserving at present, because Preservation Treatment is bad, causes the annual wastage rate of fruit 20% to 25%, and the annual wastage rate of vegetables reach 30%, the more than one hundred million tons of waste that fruits and vegetables are annual.If make good use of preservation technique, Perserving materials and corresponding facility; The income that increases is quite surprising, and relevant statistical information shows, if make China's fruits and vegetables wastage rate reduce by 3% to 5%; Can reduce 2,000,000 tons of fruit losses national every year, reduces more than 1,000 ten thousand tons of vegetables losses; If the preservation and freshness amount accounts for 15% to 20% of ultimate production, a year preservation and freshness amount is 6,000 ten thousand tons to 8,000 ten thousand tons, presses the per kilogram storage with 0.2 yuan of calculating, and the fruits and vegetables output value in the whole nation can increase by 12,000,000,000 yuan to 16,000,000,000 yuan.
Current fruit freshness preserving is mainly controlled by means of use chemistry and physical method, yet, many chemical preservatives such as Sodium Nitrite, Sodium Benzoate and SO
2In the fruit that can remain in storage, be detrimental to health, and many physical methods can't be applied owing to receive restriction such as appointed condition, operative technique.Mikrobe is to be distributed widely in natural biological group; Because kind is abundant, pathways metabolism is various; Can produce multiple biologically active substance, various vegeto-animal physiology, biochemical metabolism are had significant effects, some mikrobe has stronger antagonistic action to various pathogenic micro-organisms.Therefore; Mikrobe is applied to the fresh-keeping of longan fruit; Great potential is arranged, Gutter in nineteen fifty-three reported first subtilis citrus fruit is adopted the back pathogenic bacteria antagonistic action is arranged, at present (Huang have registered with the antibiontses of 2 kinds of control fruit postharvest diseases in U.S. etc.; 1995), but do not see short genus bacillus as the application of producing longan mikrobe preserving fungus agent.
[summary of the invention]
Technical problem to be solved by this invention is to provide a kind of new short Bacillus strain FJAT-0809-GLX (Brevibacillus brevis); This short Bacillus strain FJAT-0809-GLX was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 08 27th, 2010; The address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Deposit number is CGMCC No.4115, and adopts the fermented liquid of this new bacterial strain to obtain a kind ofly can to carry out fresh-keeping mikrobe preserving fungus agent to longan.
The present invention solves the problems of the technologies described above through following technical scheme:
This laboratory is separated from the watermelon rhizosphere soil of Yongtai County, Fujian Province and is screened the bacterial strain that obtains having antagonistic action; And this bacterial strain is delivered to Institute of Microorganism, Academia Sinica its physicochemical property etc. is identified, identify that finally it is the bacterial strain of bacillus brevis Pseudomonas.
Further, adopt the fermented liquid of this new bacterial strain to prepare and a kind ofly can carry out fresh-keeping mikrobe preserving fungus agent to longan, the concrete steps of its preparation method are:
(1) activation of bacterial strain: with transfering loop short Bacillus strain FJAT-0809-GLX is lined on the strain cultures, and in constant incubator, cultivate 24h, and culture temperature is 30 ℃;
(2) preparation seed liquor: single colony inoculation of the short Bacillus strain FJAT-0809-GLX that step (1) is obtained and is placed in the constant-temperature shaking shaking table and cultivates 24h, 30 ℃ of temperature, rotating speed 180rpm/min in the seed culture medium of 100ml;
(3) preparation liquid fermentation liquid: the seed liquor that step (2) is obtained by 10% culture transferring amount changes in the sterilized fermention medium cultivates; 30 ℃ of temperature; Rotating speed 180rpm/min; And in culturing process, get an amount of fermented liquid at set intervals and detect its pH value and spore quantity, when its spore count reaches 10 * 10
8Then obtain required liquid fermentation liquid during/mL;
(4) preparation of preserving fungus agent: mix the back autoclaving to NaCl and agar powder according to 10: 1 quality proportioning; When treating that temperature is cooled to 40-50 ℃, the liquid fermentation liquid that step (3) is obtained and this mixed solution can obtain said longan mikrobe preserving fungus agent with 4: 1 ratio mixing.
Further, the component of said strain cultures: beef extract 0.3%, peptone 0.5%, glucose 1%, agar powder 1.7%, zero(ppm) water preparation, pH7.0;
The component of said seed culture medium: starch 1%, beef extract 0.5%, peptone 0.3%, sucrose 1%, yeast powder 0.5%, CaCl
20.5%, zero(ppm) water preparation, pH7.0;
The component of said fermention medium: starch 0.8%, soybean cake powder 3%, peptone 0.2%, sucrose 2%, CaCl
20.5%, skimmer 0.17%, zero(ppm) water preparation, pH7.0.
Beneficial effect of the present invention is: a kind of new short Bacillus strain FJAT-0809-GLX is provided; And adopt this short Bacillus strain fermented liquid preparation to carry out fresh-keeping mikrobe preserving fungus agent to longan; This microbial inoculum can reduce the rotting rate of longan, and can keep the color and luster and the taste of fruit.
[embodiment]
A kind of new short Bacillus strain FJAT-0809-GLX of the present invention separates from the watermelon rhizosphere soil of Yongtai County, Fujian Province and bacterial strain that screening obtains having antagonistic action, and the fermented liquid through this bacterial strain prepares and a kind ofly can carry out fresh-keeping mikrobe preserving fungus agent to longan.
1. the separation of this bacterial strain FJAT-0809-GLX:
(1) get watermelon rhizosphere soil 10g in the 90mL sterilized water, fully vibration back absorption 1mL carries out gradient dilution, and selecting extent of dilution for use is 10
-4, 10
-5Or 10
-6
(2) the soil diluent that step (1) is obtained is coated on the NA culture medium flat plate, places 30 ℃ to cultivate 2d down the NA culture medium flat plate then;
(3) each inoculation that step (2) cultivate is obtained is in nutrient agar, and at 30 ℃ of temperature, rotating speed 180rmin
-1Cultivate 48h under the condition, collect nutrient solution;
(4) through supplying examination pathogenic bacteria (separating the 4 strain longan saprophytic bacterias that obtain the longan of a kind of " the good fortune eye " by name of gathering from Quanzhou City, the Fujian Province institute of agricultural sciences) and adopting cup-plate method that the bacterial strain in the nutrient solution of collecting is carried out the antagonism screening; Be specially: get said nutrient solution 150 μ l in the cup of Oxford; Observe behind the cultivation 48h down at 30 ℃; And adopting sterilized water to make negative control, agricultural Vetstrep is made positive control CK, obtains antagonistic strain through observing the screening of inhibition zone size.
2. the evaluation of this bacterial strain FJAT-0809-GLX:
The antagonistic strain that is obtained is delivered to Institute of Microorganism, Academia Sinica its physicochemical property etc. is identified that the main form and the biological property that obtain this bacterial strain are following: bacterium colony is matt, and is opaque; Light yellow; Surface wettability, gramstaining is positive, and the nourishing body cell is shaft-like; About 0.7 * 2.6 μ m of size, peritrichous; Gemma is closely oval, and middle life is not expanded, and does not have other special construction.Below be its concrete Physiology and biochemistry identification of indicator:
Annotate :+expression has effect or responds;-expression no effect or reactionless
Identify that this bacterial strain is short genus bacillus (Brevibacillus brevis) after combining 16S rDNA sequence to compare above-mentioned each index.
3. the concrete preparation process of longan mikrobe preserving fungus agent is:
(1) activation of short Bacillus strain FJAT-0809-GLX: with transfering loop short Bacillus strain FJAT-0809-GLX is lined on the strain cultures, and in constant incubator, cultivate 24h, and culture temperature is 30 ℃;
(2) preparation seed liquor: single colony inoculation of the short Bacillus strain FJAT-0809-GLX that step (1) is obtained and is placed in the constant-temperature shaking shaking table and cultivates 24h, 30 ℃ of temperature, rotating speed 180rpm/min in the seed culture medium of 100ml;
(3) preparation liquid fermentation liquid: the seed liquor that step (2) is obtained by 10% culture transferring amount changes in the fermention medium of sterilization and cultivates; 30 ℃ of temperature; Rotating speed 180rpm/min; And in culturing process, get an amount of fermented liquid at set intervals and detect its pH value and blood cell quantity, when its spore count reaches 10 * 10
8Then obtain required liquid fermentation liquid during/mL;
(4) preparation of preserving fungus agent: mix the back autoclaving to NaCl and agar powder according to 10: 1 quality proportioning; When treating that temperature is cooled to 40-50 ℃, the liquid fermentation liquid that step (3) is obtained and this mixed solution can obtain said longan mikrobe preserving fungus agent with 4: 1 ratio mixing.
Wherein, the component of strain cultures: beef extract 0.3%, peptone 0.5%, glucose 1%, agar powder 1.7%, sterilized water preparation, pH7.0;
The component of seed culture medium: starch 1%, beef extract 0.5%, peptone 0.3%, sucrose 1%, yeast powder 0.5%, CaCl
20.5%, sterilized water preparation, pH7.0;
The component of fermention medium: starch 0.8%, soybean cake powder 3%, peptone 0.2%, sucrose 2%, CaCl
20.5%, skimmer 0.17%, sterilized water preparation, pH7.0.
In addition, the per-cent in above-mentioned each nutrient media components is mass ratio.
4. longan mikrobe preserving fungus agent is carried out fresh-keeping test:
(1) test materials
Experimental cultivar: Quanzhou City, Fujian Province longan main breed---good fortune eye
(2) TP
(time of spraying is that longan is gathered afternoon of previous day or at dusk to the storage chemical spraying method; Weather is for fine): experimental group and control group are set, two groups choose respectively 5 strain growing ways evenly and be born in sustained height longan fruit spray, after 50 times of the longan mikrobe preserving fungus agent dilutions experimental group is sprayed; And control group adopts clear water to spray for contrast; Longan after then two groups being gathered is divided in the agricultural large bamboo or wicker basket, puts down gently, places indoor preservation; And room temperature 28-32 ℃ with down storage of relative humidity 60-74%, the laggard fruit rate and soluble solid content and fruit colour of acting charitably investigated in 5 days.
(3) test-results
As shown in table 1, in room temperature storage after 5 days, from good fruit rate, the good fruit rate of control group has only 47.46%, and the good fruit rate of the experimental group of handling through longan mikrobe preserving fungus agent reaches 83.33%; See that from the percentage of thresh percentage of thresh of experimental group is minimum has only 12.96%, and the fruit rate of taking off of control group reaches 69.70% height; Soluble solid content and control group that longan mikrobe preserving fungus agent is handled the back longan fruit are more or less the same; Control group is 16.50%; The soluble solid content that longan mikrobe preserving fungus agent is handled back (being experimental group) is 16.40%; Explain that great changes will take place for the quality of fruit through after the processing of longan mikrobe preserving fungus agent.
Table 1 longan mikrobe preserving fungus agent is to the fresh-keeping effect of longan
To sum up, the longan mikrobe preserving fungus agent that the present invention utilizes the fermented liquid of short Bacillus strain FJAT-0809-GLX to prepare gained not only can reduce the rotting rate of longan, and can keep the color and luster and the taste of fruit.
Claims (3)
1. new short Bacillus strain; It is characterized in that: said bacterial strain is short Bacillus strain FJAT-0809-GLX (Brevibacillus brevis); Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 08 27th, 2010, deposit number is CGMCC No.4115.
2. longan mikrobe preserving fungus agent, it is characterized in that: the concrete steps of its preparation method are:
(1) activation of the short Bacillus strain FJAT-0809-GLX described in the claim 1: the short genus bacillus FJAT-0809-GLX described in the claim 1 is lined on the strain cultures with transfering loop; And in constant incubator, cultivate 24h, and culture temperature is 30 ℃;
(2) preparation seed liquor: single colony inoculation of the short Bacillus strain FJAT-0809-GLX that step (1) is obtained and is placed in the constant-temperature shaking shaking table and cultivates 24h, 30 ℃ of temperature, rotating speed 180rpm/min in the seed culture medium of 100ml;
(3) preparation liquid fermentation liquid: the seed liquor that step (2) is obtained by 10% culture transferring amount changes in the fermention medium of sterilization and cultivates; 30 ℃ of temperature; Rotating speed 180rpm/min; And in culturing process, get an amount of fermented liquid at set intervals and detect its pH value and spore quantity, when its spore count reaches 10 * 10
8Then obtain required liquid fermentation liquid during/mL;
(4) preparation of preserving fungus agent: mix the back autoclaving to NaCl and agar powder according to 10: 1 quality proportioning; When treating that temperature is cooled to 40-50 ℃, the liquid fermentation liquid that step (3) is obtained and this mixed solution can obtain said longan mikrobe preserving fungus agent with 4: 1 ratio mixing.
3. like the said a kind of longan mikrobe preserving fungus agent of claim 2, it is characterized in that: the component of said strain cultures: beef extract 0.3%, peptone 0.5%, glucose 1%, agar powder 1.7%, zero(ppm) water preparation, pH7.0;
The component of said seed culture medium: starch 1%, beef extract 0.5%, peptone 0.3%, sucrose 1%, yeast powder 0.5%, CaCl
20.5%, zero(ppm) water preparation, pH7.0;
The component of said fermention medium: starch 0.8%, soybean cake powder 3%, peptone 0.2%, sucrose 2%, CaCl
20.5%, skimmer 0.17%, zero(ppm) water preparation, pH7.0.
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| CN102559786A (en) * | 2012-01-20 | 2012-07-11 | 福建省农业科学院农业生物资源研究所 | Method for preparing ethylparaben from Brevibacillus brevis strain |
| CN103266100A (en) * | 2013-04-27 | 2013-08-28 | 福建省农业科学院农业生物资源研究所 | Ultrasonic disruption method for intracellular substances in brevibacillus brevis strain |
| CN103275889A (en) * | 2013-04-27 | 2013-09-04 | 福建省农业科学院农业生物资源研究所 | Ethylparaben-producting fermentation medium of brevibacillus brevis strain and fermentation method |
| CN103267811A (en) * | 2013-04-27 | 2013-08-28 | 福建省农业科学院农业生物资源研究所 | High performance liquid chromatographic detection method for ethylparaben in fermentation broth of brevibacillus brevis strain |
| CN103266155B (en) * | 2013-05-21 | 2016-05-04 | 湖南农业大学 | The method of high performance liquid chromatography separation and purification endophytic bacterium bacterial strain antibacterial peptide |
| CN104894006B (en) * | 2015-05-12 | 2017-09-15 | 贵阳医学院 | New Brevibacillus brevis bacterial strain, cultural method and its application |
| CN105166397B (en) * | 2015-07-24 | 2020-06-26 | 福建省农业科学院农业生物资源研究所 | Brevibacillus brevis microecological preparation |
| CN108783046A (en) * | 2018-05-09 | 2018-11-13 | 福建省农业科学院 | A kind of application process that probiotics is raised pigs on the big column of fermentation bed |
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| CN101053342A (en) * | 2007-05-10 | 2007-10-17 | 南京师范大学 | Bio-preserving fungus agent and its preparation method |
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