CN108048379B - Symbiotic bacillus pumilus RFKF6 strain of red-heart kiwi fruit and application thereof - Google Patents

Symbiotic bacillus pumilus RFKF6 strain of red-heart kiwi fruit and application thereof Download PDF

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CN108048379B
CN108048379B CN201810137962.XA CN201810137962A CN108048379B CN 108048379 B CN108048379 B CN 108048379B CN 201810137962 A CN201810137962 A CN 201810137962A CN 108048379 B CN108048379 B CN 108048379B
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王思扬
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Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a symbiotic bacillus pumilus RFKF6 strain of red-heart kiwi fruits and application thereof. The preservation number of the Bacillus pumilus RFKF6 strain is CGMCC No. 14873. The Bacillus pumilus RFKF6 strain has significant antibacterial activity, can produce pectinase and epsilon-polylysine with high activity, can be applied to the preparation of bacteriostat, pectinase and epsilon-polylysine, and can also be applied to industrial production in the form of live bacteria instead of the bacteriostat, the pectinase and the epsilon-polylysine.

Description

Symbiotic bacillus pumilus RFKF6 strain of red-heart kiwi fruit and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to red-heart kiwi fruit symbiotic bacillus pumilus (Bacillus pumilus) RFKF6 strain and application thereof.
Background
Shewanella (Shewanella) is a type of marine bacteria discovered in recent years and has been formally named in 1985. On the one hand, Shewanella is utilized by humans for its excellent bioremediation function and great potential as a biofuel. On the other hand, Shewanella putrefaciens and the like are also main causes of the putrefaction of cold water fish, and further, are serious threats to human health and aquaculture as pathogenic bacteria. However, the focus of research is mainly on the advantages of energy development, and the research on the harmful aspects of human-fish co-morbidities is still in the beginning stage.
At present, the prevention and treatment of Shewanella are mostly carried out by adopting traditional methods such as chemical synthesis medicines, antibiotics and the like, so that the drug resistance of bacteria is increased, the ecological imbalance of microorganisms is caused, the double infection is generated, the antibiotics are remained in organisms, chronic poisoning and the like can be caused by long-term intake of human bodies, and the sustainable development of the aquaculture industry is influenced; the repeated blockage of the export of aquatic products in China due to the abuse of antibiotics causes great loss to the water supply industry.
Disclosure of Invention
The invention aims to inhibit Shewanella, and antagonistic bacteria is separated and screened from red-heart kiwi fruit, so that a Bacillus pumilus RFKF6 strain is finally obtained. The Bacillus pumilus RFKF6 strain has remarkable Shewanella bacteriostatic activity, and can produce high-activity pectinase and epsilon-polylysine. The Bacillus pumilus RFKF6 strain can be used for preparing bacteriostatic agent, pectinase and epsilon-polylysine, and can also be used for industrial production in the form of viable bacteria instead of bacteriostatic agent, pectinase and epsilon-polylysine.
The invention provides a symbiotic bacillus pumilus RFKF6 strain of red-heart kiwi fruit, the preservation number of which is CGMCC No. 14873.
The invention provides a culture method of the bacillus pumilus RFKF6, which comprises the following steps: inoculating the Bacillus pumilus RFKF6 in a culture medium, and culturing at 25-40 ℃ for 10-72 h.
The invention provides application of the bacillus pumilus RFKF6 strain in non-therapeutic bacteriostasis.
Preferably, the inhibiting comprises inhibiting Shewanella.
The invention provides application of the Bacillus pumilus RFKF6 strain in preparing a medicament for treating Shewanella infection diseases.
The invention provides application of the bacillus pumilus RFKF6 strain in preparation of epsilon-polylysine.
The invention provides application of the bacillus pumilus RFKF6 strain in preparation of pectinase.
Has the advantages that:
the invention provides a symbiotic bacillus pumilus RFKF6 strain of red kiwi fruit, and the preservation number of the strain is CGMCC No. 14873. The metabolite of the bacillus pumilus BFKF6 strain has strong bacteriostatic activity, and especially has a remarkable inhibitory effect on Shewanella, the diameter of the inhibition zone reaches 17mm, and the diameter of the inhibition zone of 75% ethanol serving as a positive control is only 8 mm. The Bacillus pumilus BFKF6 strain and its metabolite can be used for food preservation, preservation and medicine preparation for treating Shewanella infection diseases.
The bacillus pumilus RFKF6 can also produce high-activity pectinase, can be applied to the preparation of the pectinase, and can also be applied to the food fields of fruit juice squeezing clarification, fruit wine quality improvement and the like and the medicine textile fields of fabric refining, bleaching, degumming and the like in a live bacterial form instead of the pectinase.
The bacillus pumilus RFKF6 can also produce epsilon-polylysine, can be applied to the preparation of the epsilon-polylysine, and can also be applied to industries related to the epsilon-polylysine by replacing the epsilon-polylysine in a live bacterial mode.
Biological preservation Instructions
Bacillus pumilus (Bacillus pumilus) RFKF6 is deposited in China general microbiological culture Collection center (CGMCC) at 11/13 of 2017 at the microbial research institute of China academy of sciences, Beijing, with the collection number of CGMCC No. 14873.
Detailed Description
The invention aims at inhibiting Shewanella, and antagonistic bacteria is separated and screened from red-heart kiwi fruit to finally obtain a Bacillus pumilus RFKF6 strain. The red-heart kiwi symbiotic bacillus pumilus RFKF6 strain has the preservation number of CGMCC No.14873, the gram reaction is negative, the spore colony is characterized by being circular, small, smooth in edge, opaque and white, and has remarkable Shewanella bacteriostatic activity and can produce high-activity pectinase and epsilon-polylysine.
The invention provides a culture method of the bacillus pumilus RFKF6 strain, which comprises the steps of inoculating the bacillus pumilus RFKF6 into a culture medium, and culturing for 10-72 hours at 25-40 ℃. The present invention is not particularly limited with respect to the type and source of the medium, and the complete medium, beef extract peptone medium, pectin agar medium or M conventionally used in the art for bacterial culture3G culture medium can be used; in the invention, if the aim of propagation is to culture, the culture medium is preferably a complete culture medium, and the culture temperature is preferably 27-33 ℃, and more preferably 30 ℃; the culture time is preferably 24-48 h, and more preferably 30 h.
The invention provides application of the Bacillus pumilus RFKF6 strain in non-therapeutic bacteriostasis, which comprises food fresh-keeping, medical health care instrument bacteriostasisEtc., preferably comprising inhibition of shewanella. The Bacillus pumilus RFKF6 live bacteria (final concentration 10)3cfu/mL) or metabolite epsilon-polylysine thereof
Figure BDA0001576822860000031
When the preservative is added into food to be preserved, a good bacteriostatic effect can be achieved, and further good food fresh-keeping and preservative functions can be achieved.
The invention provides application of the Bacillus pumilus RFKF6 strain in preparing a medicament for treating Shewanella infection diseases. The metabolite epsilon-polylysine of the Bacillus pumilus RFKF6
Figure BDA0001576822860000032
Can be prepared into a medically acceptable external preparation form and can achieve the obvious effect of inhibiting Shewanella when being applied to the infected part.
The invention also provides application of the bacillus pumilus RFKF6 strain in preparation of epsilon-polylysine and/or pectinase. Inoculating the Bacillus pumilus RFKF6 into a fermentation culture medium for culture to obtain a fermentation liquid; and (3) separating and purifying the culture solution to obtain epsilon-polylysine and/or pectinase. In the invention, the separation and purification method adopts the conventional epsilon-polylysine/pectinase separation and purification method in the field to separate and purify epsilon-polylysine/pectinase from fermentation liquor.
The symbiotic brevibacillus rhynchophyllus strain RFKF6 of Actinidia polygama as provided by the present invention and its application are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Acquisition of Bacillus pumilus RFKF6 Strain having high Shewanella inhibitory Activity
a. Washing clean and fresh red-heart kiwi fruits under running water to remove dust on the surface, sequentially and respectively sterilizing with 75% ethanol and 2% sodium hypochlorite for 5min under a sterile environment, repeatedly washing with sterile water, peeling with sterile scissors on a superclean bench, cutting pulp into small blocks with side length of 1cm, and placing 5 sections on each culture dish with complete culture medium; culturing at constant temperature of 27 ℃.
b. In a sterile environment, colonies on the plates are respectively picked by an inoculating needle and repeatedly streaked until pure strains are generated and stored.
c. And (3) detection of antibacterial activity: shewanella (Shewanella) uses beef extract peptone medium. Inoculating the strain into beef extract peptone liquid culture medium, and performing shake culture for 24h as indicator bacteria. Taking the indicator bacterium liquid (OD) with a pipette6000.9), uniformly coating 100 mu L of the extract on a beef extract peptone solid culture medium by using a sterile triangular coating rod, dividing the beef extract peptone solid culture medium culture dish into six areas outside the bottom of the culture dish by using a marker pen, respectively placing a filter paper sheet in each area, respectively transferring 10 mu L of fermentation broth samples of the endophytic bacterial strains (pure bacterial strains) of the red-heart kiwifruit by using a liquid transfer gun, dripping the fermentation broth samples into the center of the filter paper sheet, and placing the filter paper sheet in an incubator at 37 ℃ for culturing for three days. Each pure strain was treated 3 times repeatedly, with the positive control being 75% alcohol and the negative control being a blank liquid medium. Observing the growth condition of the indicator bacteria, and determining the diameter of the inhibition zone. Wherein the diameter of the inhibition zone of the fermentation liquor of the RFKF6 strain to Shewanella is 17mm, which is obviously higher than the inhibition activity of other strains and positive control 75% ethanol (the inhibition diameter is only 8 mm); the RFKF6 strain was identified by using the 16sRNA molecule and was determined to be Bacillus pumilus, and the colony characteristics were circular, small, smooth-edged, opaque and white.
Example 2
RFKF6 preservation effect verification experiment
a. The RFKF strain was cultured in a liquid medium of complete medium with shaking at 30 ℃ for 30 hours. Taking zymophyte liquid, centrifuging, respectively collecting thallus and collecting fermentation supernatant, and suspending thallus in sterile water to final concentration of 103cfu/mL, and filtering the strain fermentation supernatant with a 0.2 μm filter membrane to obtain an aseptic fermentation supernatant.
b. Respectively spraying the bacterial suspension and the sterile fermentation supernatant on the surface of the small yellow croaker body (blank control without spraying a sample), respectively bagging, and refrigerating at 4 ℃. The total number of bacteria and pH were measured at 0, 2 and 4d, respectively.
The results show that the small yellow croakers of each group have increased storage timeThe total number of bacteria is increased, but the increase of the thallus suspension and the small yellow croaker treated by the sterile fermentation supernatant is slow, and the total number of the bacterial colonies of the control group exceeds that of the bacteria of the first-grade product by 10 at the 4 th day6CFU/g, and the bacterial suspension and the small yellow croaker treated by the sterile fermentation supernatant still do not exceed 104CFU/g; the pH value of each group rose from day 2, but the increase in pH value of the small yellow croakers treated with the cell suspension and the sterile fermentation supernatant rose slowly, and on day 4, the pH value of the control group was 6.8 and the pH value of the treatment groups were 6.2 and 6.3, respectively.
Example 3
Verification experiment for producing pectinase by bacillus pumilus RFKF6
a. Washing clean and fresh red-heart kiwi fruits under running water to remove dust on the surface, sequentially and respectively sterilizing with 75% ethanol and 2% sodium hypochlorite for 5min under a sterile environment, repeatedly washing with sterile water, peeling with sterile scissors on a superclean bench, cutting pulp into small blocks with side length of 1cm, and placing 5 sections on each culture dish with complete culture medium; culturing at constant temperature of 27 ℃.
b. In a sterile environment, colonies on the plates are respectively picked by an inoculating needle and repeatedly streaked until pure strains are generated and stored.
c. Screening of pectinase producing bacteria test: 15mL of pectin agar medium (formula: K) was poured into each plate2HPO41g，MgSO40.5g，NaNO33g，FeSO4·7H20.01g of O, 2g of pectin, 17g of agar powder and distilled water to a constant volume of 1000mL), standing until the culture medium is solidified, selecting strains on the slant culture medium, inoculating the strains on the pectin culture medium, and culturing in a constant-temperature incubator. When the pectinase producing bacteria are screened, Congo red dyeing liquid (1mg/mL) is added into a culture dish for dyeing for about 40min, then the Congo red is poured out, a small amount of distilled water is used for slightly removing Congo red on the surface, then the distilled water is added for standing and decoloring for 10min, and the result is judged according to whether a remarkable crimson hydrolysis ring is generated or not by observing light. As a result, a clear yellowish hydrolysis ring appears around RFKF6 colony, and the ratio of the hydrolysis ring to the colony is 5.
Example 4
Enzyme activity assay
a. Inoculating the strain RFKF6 into complete culture medium, culturing at constant temperature of 30 deg.C for 30h, centrifuging at 5000g, and collecting supernatant as crude enzyme solution;
measuring enzyme activity of fermentation liquor by using a DNS colorimetric method: a, B two tubes were taken and 0.5g/mL pectin substrate 1.5mL and acetic acid-sodium acetate buffer 2.5mL were added. Adding 1mL of crude enzyme solution into tube A, shaking up, and reacting in water bath at 48 ℃ for 30 min; after the reaction, 1mL of the crude enzyme solution was added to tube B, and the two tubes were quickly placed in a boiling water bath simultaneously and heated for 5min to terminate the reaction. After cooling, respectively transferring A, B reaction solution 2mL into another two test tubes, then adding distilled water 2mL and DNS reagent 3mL, shaking up, heating in boiling water bath for 5min, cooling and fixing the volume to 25 mL. And (3) taking the reaction solution B as a reference, adjusting zero, measuring the light absorption value of the tube A at 540nm, and calculating the enzyme activity by referring to a glucose standard curve.
The results show that: the enzyme activity of the crude enzyme solution is 1225U/mL.
Example 5
a. Washing clean and fresh red-heart kiwi fruits under running water to remove dust on the surface, sequentially and respectively sterilizing with 75% ethanol and 2% sodium hypochlorite for 5min under a sterile environment, repeatedly washing with sterile water, peeling with sterile scissors on a superclean bench, cutting pulp into small blocks with side length of 1cm, and placing 5 sections on each culture dish with complete culture medium; culturing at constant temperature of 27 ℃.
b. In a sterile environment, colonies on the plates are respectively picked by an inoculating needle and repeatedly streaked until pure strains are generated and stored.
c. Qualitative detection test of epsilon-polylysine product:
① Each plate was poured with 15mL of Epsilon-polylysine primary screening medium (formulation of the Epsilon-polylysine primary screening medium: 10g glycerol, 0.1g yeast extract, NaH)2PO40.68g，KH2PO40.25g，MgSO4·7H2O 0.25g，(NH4)2SO40.66g，ZnSO4·7H2O 0.05g，FeSO4·7H2O0.01g, 140mL trace mineral solution, 0.02g methylene blue, K2Cr7O70.05g and agar powder 18g, and the volume is determined to 1000mL by double distilled water), standing until the culture medium is solidified, dipping a small amount of separated and purified strain liquid culture medium, connecting the strain liquid culture medium to an epsilon-polylysine primary screening culture medium, placing the culture medium in a 30 ℃ constant temperature incubator for culture, judging the result according to the generation and the size of a colorless transparent ring, observing an obvious transparent ring around the RFKF6 bacterial colony, and preliminarily showing that the strain has epsilon-polylysine.
② taking 1mL of M3G fermentation medium (the formula of the M3G fermentation medium is (NH)4)2SO410g, 50g of glucose, 5g of yeast extract and MgSO4·7H2O 0.5g，ZnSO4·7H2O 0.04g，FeSO4·7H2O 0.03g，K2HPO40.8g，KH2PO41.36g, distilled water to 1000mL), adding 0.5mL of bismuth potassium iodide solution, oscillating for reaction, placing in a constant temperature incubator at 30 ℃ for reaction for 30min, observing whether a precipitate is generated, and observing whether a large amount of brick red precipitates appear in RFKF6 strain fermentation liquor, which also indicates that the strain generates epsilon-polylysine.
③ A large amount of brick-red precipitate appeared in the fermentation broth of RFKF6 strain, which was further indicated by the fact that the strain was produced by epsilon-polylysine, by adding 1mL of methyl orange solution to 1mL of the fermentation supernatant of M3G fermentation medium of the strain, shaking and reacting at 30 ℃ for 30 min.
d. Quantitative detection test of epsilon-polylysine product: taking 2mL of strain fermentation liquor, and centrifuging for 15min at 4000 r/min; 0.1mL of the centrifuged supernatant was taken out and added to a test tube, and 1.9mL of a phosphate buffer (0.1mol/L) and 2mL of a methyl orange solution (1mmol/L) were sequentially added thereto, and after the mixture was placed in a constant temperature incubator at 30 ℃ for reaction for 30min, the mixture was centrifuged again at 4000r/min for 15 min. Taking 1mL of the centrifuged supernatant, adding 19mL of physiological saline for diluting by 20 times, taking 1mL of the supernatant of the M3G culture medium, adding 19mL of physiological saline for diluting by 20 times to serve as a reference solution (only methyl orange solution is changed into deionized water, other steps are consistent), and determining OD465And calculating the mass concentration of the epsilon-polylysine in the fermentation broth according to the epsilon-polylysine standard curve, and obtaining the result of the production of the epsilon-polylysine by the RFKF6 bacteriaThe amount reaches 232.2 mg/L.
Example 6
Preparation of epsilon-polylysine and verification of bacteriostatic effect
Preparation of epsilon-polylysine: culturing the strain RFKF6 in a liquid complete culture medium for 30H, centrifuging the fermentation liquid for 10min at 5000r/min, adjusting the pH of the supernatant to 8.5 by NaOH, then centrifuging for 10min at 12000r/min, adsorbing the supernatant by cation (H +) exchange resin (D152), washing and eluting by 0.2mol/L acetic acid and 0.1mol/L hydrochloric acid respectively, neutralizing the eluate to pH 6.5 by NaOH, centrifuging for 10min at 12000r/min, transferring the supernatant into a dialysis bag 3000 for dialysis for 2D, and freeze-drying to powder.
The preservation effect of epsilon-polylysine is as follows: formulating the above powder to final concentration
Figure BDA0001576822860000081
The solution of (4) is sprayed on the surface of the small yellow croaker body (blank control does not spray a sample), and the small yellow croaker body is respectively bagged and refrigerated at 4 ℃. The total number of bacteria was determined at 0, 2 and 4d samples, respectively. The results show that the total number of bacteria of each group of the small yellow croakers increases along with the increase of the storage time, but the increasing range of the small yellow croakers treated by the epsilon-polylysine solution is slow, and the total number of bacterial colonies of the control group exceeds that of the bacteria of the first-class product by 10 days at the 4 th day5CFU/g, and 10 for Epsilon-polylysine solution treated little yellow croaker3CFU/g。
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (6)

1. The symbiotic brevibacillus choshinensis RFKF6 strain of red-heart kiwifruit has a preservation number of CGMCC No. 14873.
2. The method for culturing the strain of Bacillus pumilus RFKF6 of claim 1, wherein: inoculating the Bacillus pumilus RFKF6 strain in a culture medium, and culturing at 25-40 ℃ for 10-72 h.
3. The use of the Bacillus pumilus RFKF6 strain of claim 1 for non-therapeutic bacteriostasis, i.e., inhibition of Shewanella.
4. The use of the Bacillus pumilus RFKF6 strain of claim 1 in the manufacture of a medicament for the treatment of Shewanella infectious disease.
5. The use of the strain of bacillus pumilus RFKF6 according to claim 1 for the preparation of epsilon-polylysine.
6. The use of the strain of Bacillus pumilus RFKF6 of claim 1 for preparing pectinase.
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CN1480529A (en) * 2003-06-18 2004-03-10 江南大学 High yield fungus for alkali pectinase and screen method as well as ferentation method for producing alkali pectinase by using the strain
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Publication number Priority date Publication date Assignee Title
CN1480529A (en) * 2003-06-18 2004-03-10 江南大学 High yield fungus for alkali pectinase and screen method as well as ferentation method for producing alkali pectinase by using the strain
CN104770464A (en) * 2015-04-24 2015-07-15 北京农学院 Low-temperature storage method for egg liquid

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