CN115873757A - Alteromonas mylittae and application thereof - Google Patents
Alteromonas mylittae and application thereof Download PDFInfo
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- CN115873757A CN115873757A CN202211200242.6A CN202211200242A CN115873757A CN 115873757 A CN115873757 A CN 115873757A CN 202211200242 A CN202211200242 A CN 202211200242A CN 115873757 A CN115873757 A CN 115873757A
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Abstract
The invention relates to the technical field of microorganisms, and particularly relates to alteromonas mylittae and application thereof. The invention separates and obtains Alteromonas maydis (Alteromonas macleodii) QZ9-9 from large-scale seaweed gracilaria, and the Alteromonas maybefaciens QZ is preserved in China center for type culture collection (CGMCC) in 23 months 3 in 2022 with the preservation number of CGMCC No. 245668. Compared with the existing alteromonas mellea, the strain can fully degrade agar into agar oligosaccharides with the molecular weight of less than 3kDa, and the obtained agar oligosaccharides have obvious antioxidation.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to alteromonas mylittae and application thereof.
Background
Agar is the main component of macroalgae seaweed gel, not only plays a role of a bracket in seaweed bodies, but also is a bioactive substance with the greatest application prospect at present. However, agar polysaccharide has problems of high viscosity, low water solubility and difficult absorption, and is greatly limited in practical application, which has become an important bottleneck for high-value utilization of macroalgae.
Compared with agar polysaccharide, the agar oligosaccharide which is a straight chain or branched chain compound with the polymerization degree of 2-10 and is obtained by degrading the agar polysaccharide has the advantages of rich source, various varieties, stable property, wide application, unique activity, small molecular weight, easy water solubility, no antigenicity and the like, and has various medical health-care effects.
The biodegradation method is a safer, more efficient and more environment-friendly agar oligosaccharide preparation method at present. The macromolecular agar polysaccharide is degraded into micromolecular oligosaccharide fragments, the preparation process is mild and efficient, and meanwhile, the method has the advantages that the structure of the polysaccharide is not damaged, and harmful byproducts are not generated. Therefore, the biological method is gradually becoming an important means for the industrial preparation of novel agar oligosaccharides.
Abundant macroalgae resources contain agar polysaccharides with various structures, are valuable resources for developing marine polysaccharide functional foods and searching novel polysaccharide medicine lead compounds. However, when the application of agar polysaccharide resource is insufficient, the production method is extensive, and the combination of high and new technologies is lagged behind, the agar polysaccharide is only sold to foreign countries as a raw material or is extracted by simple extensive processing to be used as food or food additives. Under the background that the industrialization of marine organism innovation technology and the high-value development of products are urgently needed at present, the research on novel strains capable of degrading agar polysaccharide and the high-value-added application thereof have important significance.
Disclosure of Invention
In view of the above, the invention provides alteromonas maydis and application thereof, the strain can degrade agar into agar oligosaccharide, the degradation rate reaches 86.45%, and the obtained agar oligosaccharide has a remarkable antioxidation effect.
The invention separates and obtains a strain QZ9-9 from large-scale seaweed gracilaria, the strain is gram-negative bacillus, the bacterial colony is wet, light yellow and round, the edge is neat, aerobic bacteria can degrade agar, a hydrolysis ring is arranged on a solid culture medium, and the diameter of the hydrolysis ring is continuously enlarged along with the increase of time. The nucleotide sequence of the 16S rRNA gene of the strain is shown as SEQ ID NO:1 is shown. Identified as Alteromonas maydis (Alteromonas macleodiii) by morphology and 16s rRNA gene, and the preservation number is CGMCCNO.24569.
The invention also provides application of the alteromonas maclekii in producing agarase and preparing agar oligosaccharide.
The invention also provides a method for preparing agar oligosaccharide, which is prepared by degrading agar by using the alteromonas maidendri QZ 9-9.
In some embodiments, the method for preparing agar oligosaccharides provided by the invention specifically comprises the following steps:
step (1): inoculating alternaria mali with the preservation number of CGMCC NO. 24569 into a seed culture medium, and culturing to obtain a seed solution;
step (2): inoculating the seed solution into a liquid culture medium containing agar, culturing, and centrifuging to remove thalli to obtain a supernatant;
and (3): and (3) performing molecular interception and centrifugation on the supernatant, and collecting components with the molecular weight of less than 3 kDa.
In some embodiments, the amount of the alteromonas maydis QZ9-9 inoculated in step (1) is 2 to 2.5% (v/v); in some embodiments, the inoculation amount may specifically be 2.5% (v/v).
In some embodiments, the conditions of the culturing are: culturing at the rotation speed of 160-180rpm and the temperature of 25-30 ℃ for 12-15 h; in some embodiments, the conditions of the culturing are: the rotation speed is 180rpm, and the culture is carried out for 12 hours at 30 ℃.
In some embodiments, in the step (2), the liquid medium containing agar comprises the following components in percentage by mass:
0.12% agar, 3% NaCl, 0.5% (NH) 4 ) 2 SO 4 、0.26％K 2 HPO 4 ·3H 2 O、0.1％MgSO 4 ·7H 2 O、0.01％FeSO 4 ·7H 2 O, pH 7.5.
In some embodiments, the seed solution in step (2) is inoculated in an amount of 2 to 2.5% (v/v); in some embodiments, the inoculation amount may specifically be 2.5% (v/v).
In some embodiments, the conditions of the culturing in step (2) are: culturing at the rotation speed of 160-180rpm and the temperature of 25-30 ℃ for 50-60 h; in some embodiments, the conditions of the culturing are: the rotation speed is 180rpm, and the culture is carried out for 50h at the temperature of 30 ℃.
The invention also provides the agar oligosaccharide prepared by the method.
The invention researches the antioxidation characteristics of the agar oligosaccharides (namely crude agar oligosaccharides with the molecular weight of less than 3 kDa) prepared by the method, and finds that the DPPH clearance rate of the agar oligosaccharides at pH 6.5-7.5 is more than 80 percent, the DPPH clearance rate at the temperature of 25-30 ℃ is more than 85 percent, and the agar oligosaccharides have remarkable antioxidation effect.
Based on the effects, the invention also provides the application of the agar oligosaccharide in preparing antioxidant products.
The invention separates and obtains Alteromonas maydis (Alteromonas macleodii) QZ9-9 from large-scale seaweed gracilaria, and the Alteromonas maybefaciens QZ is preserved in China center for type culture collection (CGMCC) in 23 months 3 in 2022 with the preservation number of CGMCC NO. 245668. The strain can fully degrade agar into agar oligosaccharide with the molecular weight less than 3kDa, and the obtained agar oligosaccharide has a remarkable antioxidation effect.
Description of biological preservation
QZ9-9, taxonomy name: alteromonas maytansi marclodii was deposited at the general microorganism center of the China Committee for culture Collection of microorganisms at 23.3.2022.3.23.the accession number of the institute of microbiology, china academy of sciences, no. 3 of West Lu 1 of the Beijing area of the republic of oriented Yang, and CGMCC No. 245668.
Drawings
FIG. 1 is a strain morphology diagram;
FIG. 2 is a gram stain of the strain;
FIG. 3 is a scanning electron micrograph of the strain;
FIG. 4 shows the change of hydrolysis rings of strains degrading agar at different time periods;
FIG. 5 is a phylogenetic tree diagram of strain QZ 9-9;
fig. 6 is the oxidation resistance of agar oligosaccharides less than 3 kDa;
fig. 7 is a graph of the effect of temperature on the oxidation resistance of agar oligosaccharides;
fig. 8 is a graph of the effect of pH on the oxidation resistance of agar oligosaccharides;
FIG. 9 shows the results of comparison of the antioxidant properties of agar oligosaccharides produced by strain QZ9-9 and strain QZ 8-6.
Detailed Description
The invention discloses alteromonas maydis and application thereof, and can be realized by appropriately improving process parameters by taking the contents of the text as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention is further illustrated by the following examples:
example 1 screening and characterization of Alternaria mylittae (Alteromonas macleodii) QZ9-9
1.1 culture Medium
(1) The agar liquid culture medium comprises the following components in percentage by mass: 0.12% agar, 3% NaCl, 0.5% (NH) 4 ) 2 SO 4 、0.26％K 2 HPO 4 ·3H 2 O、0.1％MgSO 4 ·7H 2 O、0.01％FeSO 4 ·7H 2 O，pH 7.5。
(2) The agar solid culture medium comprises the following components in percentage by mass: 1.5% agar, 3% NaCl, 0.5% (NH) 4 ) 2 SO 4 、0.26％K 2 HPO 4 ·3H 2 O、0.1％MgSO 4 ·7H 2 O、0.01％FeSO 4 ·7H 2 O，pH 7.5。
1.2 Strain selection
1.2.1 sample Collection: collecting Hainan offshore large-scale seaweed Gracilaria, taking 5g of sample, adding 50mL of PBS buffer solution, grinding in a sterile grinder, pouring into a 100mL EP tube, shaking up and down slightly, and standing for 10min for later use.
1.2.2 Primary screening of Strain QZ 9-9: inoculating 10 μ L of the processed Gracilaria sample to a liquid culture medium containing agar as the sole carbon source, performing fermentation culture, culturing at 30 deg.C and 180rpm for 24h, and performing 3 passages.
1.2.3 rescreening of Strain QZ 9-9: and (3) spreading 3 mu L of fermentation liquor after passage on an agar solid culture medium, and after bacterial colonies grow out, carrying out multiple streaking until a single bacterial colony is separated (figure 1).
1.3 morphological characteristics of Strain QZ9-9
The strain QZ9-9 is gram-negative bacteria and bacillus, the bacterial colony is wet, light yellow and round, the edge is neat, the aerobic bacteria can degrade agar and have a hydrolysis ring on a solid culture medium (figure 1-3), and the diameter of the hydrolysis ring is continuously enlarged along with the increase of time (5% Lugol iodine solution staining) (figure 4).
1.4 molecular biological identification of Strain QZ9-9
After genome extraction of QZ9-9, 16S rDNA sequencing and identification was performed, the strain was identified as Alteromonas maytans (Alteromonas macleodii), which we named Alteromonas maytans (Alteromonas macleodii) QZ9-9 (FIG. 5).
The length of the 16S rRNA whole gene sequence is 1381bp, and the sequence is shown as SEQ ID NO:1 is shown.
Method for preparing agar oligosaccharide from 1.5 strain QZ9-9
(1) Alteromonas maydis QZ9-9 was inoculated into 2216E seed medium at an inoculum size of 2.5% (v/v), rotated at 180rpm, charged at 25%, and cultured at 30 ℃ for 12 hours to obtain a seed solution.
(2) Inoculating the seed solution into agar liquid culture medium with inoculation amount of 2.5% (agar liquid culture medium for preparing agar oligosaccharide: 0.12% agar, 3% NaCl, 0.5% (NH) 4 ) 2 SO 4 、0.26％K 2 HPO 4 ·3H2O、0.1％MgSO 4 ·7H 2 O、0.01％FeSO 4 ·7H 2 O, pH 7.5). 180rpm, culturing at 30 deg.C for 50h to obtain fermentation liquid, and centrifuging to remove thallus to obtain supernatant;
(3) And (3) obtaining crude agar oligosaccharides with the molecular weight of less than 3kDa by using an Ultra centrifuge tube through a molecular interception method for supernate.
1.6 QZ9-9 agar oligosaccharide antioxidation characteristic
1.6.1 the antioxidation of the agar oligosaccharide with less than 3kDa
The antioxidant properties of agar oligosaccharides were evaluated using DPPH free radical scavenging. A0.1 mM DPPH solution was prepared using absolute ethanol. Firstly, the antioxidant property of agar oligosaccharide with the content of less than 3kDa is considered, 50 mu L of DPPH solution is mixed with 150 mu L of agar oligosaccharide solution with the content of less than 3kDa (pH 7.0), after incubation for 60min in a dark place at 25 ℃, the absorbance of the reaction solution is measured at 517nm, and absolute ethyl alcohol is used for replacing a sample as a negative control, and the antioxidant efficiency of the agar oligosaccharide with the content of less than 3kDa is calculated. The result shows that the DPPH clearance of the agaropectin oligosaccharide with < 3kDa after molecular interception is 88 percent, which is improved by nearly 68 percent compared with the fermentation liquor with the same volume in the step (2) in the section 1.5, and the agaropectin oligosaccharide shows stronger anti-oxidation property (figure 6).
1.6.2 Effect of temperature on antioxidant Activity of agar oligosaccharides
Mixing 50 mu L of DPPH solution with 150 mu L of agar oligosaccharide solution with the purity of less than 3kDa (pH 7.0), incubating the mixture at different temperatures in the dark for 60min, measuring the absorbance of the reaction solution at 517nm, using absolute ethyl alcohol to replace a sample as a negative control, and observing the antioxidant efficiency of the agar oligosaccharide with the purity of less than 3kDa at different temperatures, wherein the result shows that the DPPH clearance rate at the temperature range of 25-30 ℃ is optimal and is all more than 87% (figure 7).
1.6.3 Effect of pH on the Oxidation resistance of agar oligosaccharides
Firstly, the pH value of the agar oligose liquid with the concentration of less than 3kDa is adjusted to 5-11. Then, 50 mu L of DPPH solution and 150 mu L of agar oligosaccharide solution with the purity of less than 3kDa are mixed, after the mixture is incubated for 60min at 25 ℃ in the dark, the absorbance of the reaction solution is measured at 517nm, absolute ethyl alcohol is used as a negative control to replace a sample, and the antioxidant efficiency of the agar oligosaccharide with the purity of less than 3kDa at different temperatures is examined, and the result shows that the DPPH clearance rate is optimal and is all more than 83% when the pH range is 6.5-7.5.
Comparative example 1
In the strain screening of example 1, the invention also obtained the strain QZ8-6, prepared agar oligosaccharides by fermentation of the strain QZ8-6 according to the method of section 1.5, and tested the antioxidant effect of the obtained agar oligosaccharides according to the method of section 1.6.1, the results are shown in fig. 9.
The result shows that the DPPH clearance rate of the agar oligosaccharide with the molecular weight less than 3kDa prepared by degrading agar by the strain QZ8-6 is far lower than that of the strain QZ9-9 of the invention and is about 14 percent, and the antioxidation effect is far lower than that of the strain of the invention.
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 (10)
1. Alteromonas maydis (Alteromonas macleodii) with the preservation number of CGMCC NO. 24569.
2. The alteromonas maydis according to claim 1, wherein the nucleotide sequence of the 16S rRNA gene is as set forth in SEQ ID NO:1 is shown.
3. Use of alteromonas macbechamii according to claim 1 or 2 in the production of agarase and the preparation of agaro-oligosaccharides.
4. A method for preparing agar oligosaccharide, which is characterized in that the agar is prepared by degrading agar by alteromonas maydis according to claim 1 or 2.
5. The method of claim 4, comprising:
step (1): inoculating alternaria mali with the preservation number of CGMCC NO. 24528 into a seed culture medium, and culturing to obtain a seed solution;
step (2): inoculating the seed solution into a liquid culture medium containing agar, culturing, and centrifuging to remove thalli to obtain a supernatant;
and (3): and (3) carrying out molecular interception and centrifugation on the supernatant, and collecting components with the molecular weight of less than 3 kDa.
6. The method according to claim 5, wherein in step (1), the inoculum size of alteromonas maydis QZ9-9 is 2-2.5% (v/v); the culture conditions are as follows: the rotating speed is 160-180rpm, and the culture is carried out for 12-15 h at the temperature of 25-30 ℃.
7. The method according to claim 5, wherein in the step (2), the agar-containing liquid culture medium comprises the following components in percentage by mass and volume:
0.12% agar, 3% NaCl, 0.5% (NH) 4 ) 2 SO 4 、0.26％K 2 HPO 4 ·3H 2 O、0.1％MgSO 4 ·7H 2 O、0.01％FeSO 4 ·7H 2 O, pH 7.5.
8. The method of claim 5, wherein in step (2), the inoculation amount of the seed solution is 2.5% (v/v); the culture conditions are as follows: the rotating speed is 160-180rpm, and the culture is carried out for 50-60 h at the temperature of 25-30 ℃.
9. Agar oligosaccharides obtainable by the process according to any one of claims 4 to 8.
10. The use of agar oligosaccharides as claimed in claim 9 in the preparation of antioxidant products.
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| CN117165490A (en) * | 2023-09-25 | 2023-12-05 | 山东电力工程咨询院有限公司 | Alteromonas from ocean and application thereof |
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| CN117165490A (en) * | 2023-09-25 | 2023-12-05 | 山东电力工程咨询院有限公司 | Alteromonas from ocean and application thereof |
| CN117165490B (en) * | 2023-09-25 | 2024-04-12 | 山东电力工程咨询院有限公司 | Alteromonas from ocean and application thereof |
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