CN111996141A - Microbacterium oxydans NA2 and application thereof - Google Patents
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Abstract
The invention relates to a Microbacterium oxydans NA2 and application thereof, wherein the preservation number of Microbacterium oxydans NA2 is CGMCC NO.18777, and the Microbacterium oxydans is a potato endophyte separated from potatoes and well grown on a YIM38 solid culture medium. Experiments prove that the toxic substance solanine can be effectively inhibited from increasing in the potato storage process by inoculating Microbacterium oxydans NA2 before the potatoes are stored, and the use safety of the potatoes is obviously improved. The invention obviously reduces the loss of the solanine content increase to the potato industry by a biological control mode, and has practical application value in the aspects of potato planting, potato storage and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to microbacterium oxydans NA2 and application thereof.
Background
The potato is one of the main grain crops at present, contains a large amount of starch, can provide abundant heat for human bodies, is rich in various proteins, amino acids, vitamins and minerals, and is beneficial to inhibiting weight gain and preventing obesity due to low fat content. At present, processed products of potatoes are continuously and comprehensively developed and utilized, such as 1, potato foods (potato chips, strips, dices, purees, puffed foods); 2. potato vermicelli, vermicelli; 3. potato starch; 4. potato flakes, and the like. In addition, the potatoes are also widely applied to the fields of textile, papermaking, chemical industry, building materials, medicine and the like.
In the process of storing the potatoes after picking, because of improper storage mode or external damage, adverse changes such as germination toxin change, rising of solanine content and the like which affect human health are easily generated, the edible safety of the potatoes is affected, the commodity value of the potatoes is reduced, and economic loss is caused. Solanine, also called solanine, which is a bioactive alkaloid contained in potatoes, has certain toxicity to human bodies, has a latency period of several minutes to several hours, and has the symptoms of tongue and pharynx paralysis, stomach burning pain, diarrhea, vomiting, fever, dizziness, mydriasis, tinnitus, excitation, language disorder, spasm and the like. Therefore, how to inhibit the solanine content in potatoes becomes a technical problem to be solved urgently, and researches on the biological control of solanine in potatoes are few.
Endophytes, as a potential resource of huge microorganisms in plant tissues, are increasingly paid more attention by researchers due to the complex and close metabolic relationship between endophytes and hosts. Endophytes include endophytes, actinomycetes, fungi, ubiquitous within the various organs and tissues of plants, which affect the host by: (1) increasing the nutrition obtained by the plant; (2) protecting plants from pathogenic bacteria and insects; (3) increasing the tolerance of a plant to stress; (4) modulating the development of the plant; (5) suppressing the growth of weeds. The endophyte lives in a special environment in a plant body for a long time and co-evolves with a host, and a mutual benefit symbiosis relationship is formed in the evolution process. Researches show that the endophyte can produce abundant and diverse secondary metabolites with pesticide activity, and has potential application and development values for preventing and treating plant diseases.
Disclosure of Invention
In order to solve at least one of the problems of the prior art, the application provides a microbacterium oxydans NA2 and application thereof.
In a first aspect, the present invention provides a Microbacterium oxydans (Microbacterium oxydans) NA2 having the following accession information: the preservation number is: CGMCC NO. 18777; the classification is named as: microbacterium oxydans of Microbacterium oxydans; the preservation unit is as follows: the institute of microorganisms of the Chinese academy of sciences; the preservation address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation date is 11 months and 1 day in 2019.
The invention separates and obtains Microbacterium oxydans NA2 by the following method:
potato (Atlantic) tubers collected from Dingxi city of Gansu province in China are subjected to surface sterilization, cut into 5mm small sections by using a sterile scalpel, placed on a separation culture substrate, cultured at 28 ℃ until a single bacterial colony appears in a sample, subjected to streak purification on a YIM38 culture medium, and subjected to point grafting purification and transfer for 2-3 times to obtain a single strain. The strain is identified to be Microbacterium oxydans (Microbacterium oxydans) and named as NA2 by performing physiological and biochemical identification and 16S rRNA (SEQ ID NO.1) gene sequence comparison according to a bacterial identification manual.
The physiological characteristics of Microbacterium oxydans (Microbacterium oxydans) NA2 are as follows: the growth on YIM38 solid medium is good, the optimal growth temperature is 28 ℃, and the optimal growth pH is 7.2. In YIM38 medium, the bacterial strain has a plate colony morphology of round, convex, smooth, moist, yellow, gram-positive.
The invention further provides a microbial inoculum comprising the Microbacterium oxydans NA 2.
In a second aspect, the present invention provides a method of improving the quality of potatoes comprising: before the potatoes are stored, the potatoes are inoculated with the microbial inoculum of the Microbacterium oxydans (Microbacterium oxydans) NA2 or 3 according to claim 1 or 2.
Further, the potatoes are stored under the condition of 100% illumination at 26-30 ℃.
Further, the microbial inoculum is a fermentation product of Microbacterium oxydans NA2, and the preparation method comprises the following steps:
inoculating the Microbacterium oxydans NA2 to a YIM38 culture medium, and culturing at pH of 7.0-7.2 and temperature of 27-29 ℃ for 48h at the rotation speed of 200r.min-1Is obtained by fermentation under the condition of (1).
Further, the YIM38 medium comprises:
4g of Glucose (Glucose), 4g of Yeast extract powder (Yeast extract), 5g of Malt extract powder (Malt extract), 1g of vitamin B (B-Vitamins), 1g of sodium chloride (Trace salt) and 1L of ultrapure water.
Further, the preparation method also comprises the following steps:
and (3) centrifugally separating supernatant of the fermentation liquor and thalli, extracting by using ethyl acetate and propanol with equal volumes respectively, performing rotary evaporation, redissolving by using 1mL of methanol, and storing at the temperature of-20 ℃.
The invention further provides the Microbacterium oxydans NA2 and application of the microbial inoculum in inhibiting synthesis of solanine.
The invention further provides the Microbacterium oxydans NA2 and application of the microbial inoculum in inhibiting synthesis of alpha-solanine and alpha-kaline.
The invention further provides the Microbacterium oxydans NA2 and application of the microbial inoculum in improving the quality of potatoes.
The invention has the following beneficial effects:
the potato endophyte provided by the invention can obviously reduce the solanine content in the potato storage process, and the inhibition rate of the solanine content in 7 days of storage reaches 36%. The invention discovers that the synthesis of alpha-solanine and alpha-kaline can be inhibited by inoculating Microbacterium oxydans (Microbacterium oxydans) NA2 to potato tubers, thereby improving the edible safety of potatoes and greatly improving the storage quality of potatoes in postharvest storage.
Drawings
FIG. 1 is a colony map of Microbacterium oxydans NA2 provided in example 1 of the present invention;
FIG. 2 is an electron microscope scan of Microbacterium oxydans NA2 provided in example 1 of the present invention;
FIG. 3 is a phylogenetic diagram of Microbacterium oxydans NA2 constructed based on the 16S rRNA gene sequence according to example 1 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
In the embodiment, the process for separating and purifying endophytes for inhibiting the increase of the total solanine content of the potatoes comprises the following steps:
1. materials: the potato variety is Atlantic, collected from Dingxi City of Gansu province;
2. and (3) disinfection: carrying out ultrasonic treatment on potato tubers by using ultrapure water for 15min, soaking in 0.01% Tween-20 for 1min, soaking in 3% sodium chlorate for 7min, soaking in 2.5% sodium thiosulfate for 9min, soaking in 75% alcohol for 3min, and washing with distilled water for 5 times, wherein 200 mu L of washing water is taken for coating an ISP2 flat plate for the third time, and then soaking in 10% sodium bicarbonate for 10 min;
3. separation: cutting potato tubers with sterilized surfaces, taking 5mm samples respectively, naturally air-drying in a super clean workbench, uniformly placing on 11 separation culture media added with potato juice, and culturing at 28 ℃ for 3 weeks;
4. culture medium: 10% concentration Nutrient Agar (NA): 0.5g of Beef extract (Beef extract), 1g of Peptone (Peptone), 1.25g of Sodium pyruvate (Sodium pyruvate), 1.25g of Betaine (Betaine), 10g of Sodium chloride (NaCl), 20.0g of Agar powder (Agar) and 1L, pH value of ultrapure water of 7.2, 1% of potato extract. Preparing potato extract: 100g of cleaned potato tubers are taken, cut into blocks, homogenized, transferred into a 1000ml conical flask, added with 500ml of distilled water, placed into 3-5 glass beads and shaken for 1h (200 r.min-1).
5. Observing and selecting single colonies with different appearance morphological characteristics, streaking and purifying on a plate on a YIM38 culture medium, carrying out point grafting purification and switching for 2-3 times, selecting a single strain, switching the purified strain to a YIM38 culture medium, and storing at 4 ℃ for later use. The pure culture obtained above was frozen in 30% glycerol at-80 ℃. And carrying out thallus morphology observation and physiological and biochemical analysis on the separated single colony, and carrying out 16S rDNA sequence analysis on the thallus of the colony or the bacterial strains with obvious difference in physiological and biochemical characteristics.
6. The obtained potato endophytic strain is identified, wherein the identification result of Microbacterium oxydans NA2 is as follows:
(1) morphological identification
In YIM38 medium, the strain had a plate colony morphology of round, convex, smooth, moist, yellow, gram-positive (FIG. 1). As shown in FIG. 2, the rod-shaped material had a diameter of 0.2 to 0.5. mu.m.times.0.6 to 1.6 μm (width. times.length).
(2) Physiological and biochemical identification
The specific experimental method for measuring physiological and biochemical indexes refers to R.E. Bukannan et al Bergey bacteria identification Manual and Dongxu Zhu et al common bacteria system identification Manual. The identification result of the physiological and biochemical indexes of the strain is as follows:
TABLE 1. Microbacterium oxydans NA2 physiological and biochemical identification results
Note: "+" represents a positive reaction and "-" represents a negative reaction.
The strain NA2 was identified as Microbacterium by combining its morphological and physiological biochemical characteristics.
(3) Molecular biological identification
16S rDNA identification: the extraction of total DNA from strain NA2 was carried out by the Chelex-100 method (Zhoudashuang et al, 2010). An appropriate amount of thallus is picked from a solid culture medium by using a sterile inoculating loop and placed in a 1.5mL sterile Eppendorf tube, 50 mu L of 5% Chelex100 solution is added, boiling water bath is carried out for 15min, after cooling to room temperature, centrifugation is carried out for 5min at 12000rpm, and supernatant is taken as a DNA template and stored at-20 ℃ for standby. The primer used in the PCR amplification of the 16S rRNA gene is synthesized by Shanghai biological engineering technology, Inc., and the sequence is as follows: 27 f: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively; 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' are provided. The PCR reaction consisted of 50. mu.L of 2. mu.L of DNA template, 25. mu.L of 1 × Easytap PCR Supermix enzyme, 27f and 1492r primers, 1.5. mu.L each, and 20. mu.L of sterile water. The reaction procedure is shown in Table 2.
TABLE 2 PCR reaction procedure
The PCR product was subjected to 1.2% agarose gel electrophoresis and then subjected to sequencing by Shanghai Bioengineering technology Ltd. The measured gene sequence (SEQ ID NO.1) was logged in an EZBiocoud database (http:// http:// www.ezbiocloud.net /) and subjected to 16S rRNA sequence alignment to obtain a sequence similarity alignment result. The phylogenetic tree was constructed by the Nei ghbor-join method in MEGA7.0, as shown in FIG. 3.
The molecular identification result shows that the strain NA2 is identified as Microbacterium oxydans (Microbacterium oxydans), is preserved in China general microbiological culture Collection center (CGMCC for short) in 11 months and 1 days in 2019, has a preservation number of CGMCC NO.18777, and is classified and named as: microbacterium oxydans of Microbacterium oxydans; the preservation unit is as follows: the institute of microorganisms of the Chinese academy of sciences; and (4) storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
Example 2
In this example, the specific flow of detecting the inhibition effect of Microbacterium oxydans (Microbacterium oxydans) NA2 on solanine in the storage process of potatoes is as follows:
1. and (3) screening the activity of inhibiting potato solanine: the selected strain is shaken for 48h under the conditions of 28 ℃ and 200r.min < -1 > by using a YIM38 culture medium. Potato samples were selected as required, and after being clearly air-dried, the sample groups were soaked in the strain broth for 10 minutes as experimental groups, negative control was blank YIM38 medium for 10 minutes, and positive control group was applied to potato tubers using common potato bud inhibitor chlorpropham (CIPC) at a manufacturing dose (0.01mg CIPC/g Potatoes). After all samples are treated, the samples are stored for 7 days at 28 ℃ under the 100% illumination condition, and the solanine is induced to generate.
3. The solanine content was determined (UPLC-QQQ-MS method). The method comprises the following steps: crushing potato, collecting homogenate 5g, adding 30ml acetic acid-ethanol (1: 10, V: V) mixed solvent, oscillating for 30min, centrifuging at 4 deg.C for 10min at 5000r.min-1, collecting supernatant 0.5ml, blowing nitrogen, redissolving with 2ml methanol, diluting by 10 times, and filtering with 0.22 μm organic filter membrane. Separating with C18 chromatographic column, eluting with acetonitrile-0.1% formic acid (30:70, V: V) at 20 deg.C under isocratic elution and flow rate of 0.3mL/min, and quantifying with triple quadrupole mass spectrometer under electrospray ionization (ESI + AJS) and MRM modes.
4. Suppression rate calculation method
Inhibition rate (negative control solanine content-experimental solanine content)/negative control solanine content.
This example gives the following results:
TABLE 3 inhibitory Effect of Microbacterium oxydans NA2 on solanine
As can be seen from the above table, the inhibition rate of Microbacterium oxydans NA2 on solanine reaches 36% after 7 days of storage, which is significantly higher than that of chlorpropham (1-methyl-3-chlorophenylcarbamate, CIPC), which is a potato bud inhibitor commonly used in the art.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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attactagcg actccgactt catgaggtcg agttgcagac ctcaatccga actgggaccg 180
gctttttggg attcgctcca cctcgcggta ttgcagccct ttgtaccggc cattgtagca 240
tgcgtgaagc ccaagacata aggggcatga tgatttgacg tcatccccac cttcctccga 300
gttgaccccg gcagtatccc atgagttccc accattacgt gctggcaaca tagaacgagg 360
gttgcgctcg ttgcgggact taacccaaca tctcacgaca cgagctgacg acaaccatgc 420
accacctgtt tacgagtgtc caaagagttg accatttctg gcccgttctc gtatatgtca 480
agccttggta aggttcttcg cgttgcatcg aattaatccg catgctccgc cgcttgtgcg 540
ggtccccgtc aattcctttg agttttagcc ttgcggccgt actccccagg cggggaactt 600
aatgcgttag ctgcgtcacg gaatccgtgg aatggacccc acaactagtt cccaacgttt 660
acggggtgga ctaccagggt atctaagcct gtttgctccc caccctttcg ctcctcagcg 720
tcagttacgg cccagagatc tgccttcgcc atcggtgttc ctcctgatat ctgcgcattc 780
caccgctaca ccaggaattc caatctcccc taccgcactc tagtctgccc gtacccactg 840
caggccggag gttgagcctc cggatttcac agcagacgcg acaaaccgcc tacgagctct 900
ttacgcccaa taattccgga taacgcttgc gccctacgta ttaccgcggc tgctggcacg 960
tagttagccg gcgctttttc tgcaggtacc gtcactttcg cttcttccct gctaaaagag 1020
gtttacaacc cgaaggccgt catccctcac gcggcgttgc tgcatcaggc ttgcgcccat 1080
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tggccggtca ccctctcagg ccggctaccc gtcgacgcct tggtgagcca ttacctcacc 1200
aacaagctga taggccgcga gcccatcccc aaccgaaaaa tctttccaga cgcagaccat 1260
gcggtcacgt cacatatcca gtattagacg ccgtttccag cgcttatccc agagtcaggg 1320
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Claims (9)
1. A Microbacterium oxydans NA2, wherein the Microbacterium oxydans NA2 has the deposit number: CGMCC NO. 18777.
2. Microbacterium oxydans NA2 according to claim 1, wherein the sequence of the 16s rDNA of Microbacterium oxydans NA2 is as shown in SEQ ID No. 1.
3. A microbial preparation comprising the Microbacterium oxydans NA2 according to claim 1 or 2.
4. A method of improving the quality of potatoes comprising:
before the potatoes are stored, the potatoes are inoculated with the microbial inoculum of the Microbacterium oxydans (Microbacterium oxydans) NA2 or 3 according to claim 1 or 2.
5. A method according to claim 4 wherein the potatoes are stored at conditions of 26 to 30 ℃ under 100% light.
6. The method as claimed in claim 4 or 5, wherein the microbial inoculum is a fermentation product of Microbacterium oxydans NA2, and is prepared by the following method:
inoculating the Microbacterium oxydans NA2 to a YIM38 culture medium, and culturing at pH of 7.0-7.2 and temperature of 27-29 ℃ for 48h at the rotation speed of 200r.min-1Is obtained by fermentation under the condition of (1).
7. Use of the micro bacillus oxydans (Microbacterium oxydans) NA2 according to claim 1 or 2 or the microbial agent according to claim 3 for inhibiting the synthesis of solanine.
8. Use of a micro bacillus oxydans NA2 according to claim 1 or 2 or a microbial inoculum according to claim 3 for inhibiting the increase of solanine content during potato storage.
9. Use of the micro bacillus oxydans NA2 according to claim 1 or 2 or the microbial inoculum according to claim 3 for improving potato quality.
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