A strain of Alteromonas mesquite and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of Alteromonas macleodii and its application. The present invention isolates a strain of Alteromonas macleodii QZ9-9 from the large seaweed Gracilaria, which was deposited in the China Center for Type Culture Collection on March 23, 2022, with a deposit number of CGMCCNO.24568. Compared with the existing Alteromonas macleodii, the strain can fully degrade agar into agar oligosaccharides with a molecular weight of less than 3kDa, and the obtained agar oligosaccharides have a significant antioxidant effect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Alteromonas mesquite and application thereof. Background Art

[0002] Agar is the main component of macroalgae alginate, which not only plays a scaffolding role in the algae, but is also the most promising class of bioactive substances. However, agar polysaccharides have problems such as high viscosity, low water solubility and poor absorption, which greatly restricts their practical application. This has become an important bottleneck for the high-value utilization of macroalgae.

[0003] Agar oligosaccharides, straight-chain or branched compounds with a degree of polymerization of 2-10 obtained by degradation of agar polysaccharide, are not only rich in sources, diverse in types, stable in properties, widely used, and unique in activity compared to agar polysaccharide, but also have significant advantages such as small molecular weight, easy solubility in water, and non-antigenicity. They have multiple medical and health care effects.

[0004] Biodegradation is currently a safer, more efficient and more environmentally friendly method for preparing agar oligosaccharides. By degrading large molecular agar polysaccharides into small molecular oligosaccharide fragments, the preparation process is gentle and efficient, and it also has the advantages of not destroying the structure of the polysaccharide and not producing harmful byproducts. Therefore, biodegradation has gradually become an important means for the industrial preparation of new agar oligosaccharides.

[0005] The rich resources of large seaweed contain agar polysaccharides with various structures, which are valuable resources for developing marine polysaccharide functional foods and finding new polysaccharide drug lead compounds. However, the current agar polysaccharide resources are underutilized, the production methods are extensive, and the combination of high-tech is backward. They are only sold abroad as raw materials or extracted by simple and extensive processing as food or food additives. In the current context of the urgent need for the industrialization of marine biological innovation technology and the development of high-value products, it is of great significance to study new strains that can degrade agar polysaccharides and their high value-added applications. Summary of the invention

[0006] In view of this, the present invention provides a strain of Alteromonas mesquite and its application, the strain can degrade agar into agar oligosaccharides with a degradation rate of 86.45%, and the obtained agar oligosaccharides have significant antioxidant effect.

[0007] The invention separates and obtains strain QZ9-9 from large seaweed Gracilaria, which is a gram-negative bacillus, has moist, light yellow, round colonies, neat edges, is an aerobic bacterium, can degrade agar, has a hydrolysis zone on a solid culture medium, and the diameter of the hydrolysis zone expands continuously with the increase of time. The nucleotide sequence of the 16S rRNA gene of the strain is shown in SEQ ID NO: 1. The strain is identified as Alteromonas macleodii through morphology and 16S rRNA gene, and its deposit number is CGMCCNO.24568.

[0008] The present invention also provides the use of the Alteromonas mesquite in producing agarase and preparing agar oligosaccharide.

[0009] The present invention also provides a method for preparing agar oligosaccharide, which is prepared by degrading agar by the Alteromonas mesquite QZ9-9 described in the present invention.

[0010] In some embodiments, the method for preparing agar oligosaccharide provided by the present invention specifically comprises:

[0011] Step (1): inoculating the Alteromonas mackeri with a deposit number of CGMCC NO.24568 into a seed culture medium, culturing, and obtaining a seed solution;

[0012] Step (2): inoculating the seed liquid into a liquid culture medium containing agar, culturing, and removing the bacterial cells by centrifugation to obtain a supernatant;

[0013] Step (3): The supernatant is subjected to molecular interception and centrifugation to collect components with a molecular weight less than 3 kDa.

[0014] In some embodiments, the inoculation amount of the Alteromonas mesquite QZ9-9 in step (1) is 2-2.5% (v / v); in some specific embodiments, the inoculation amount can be specifically 2.5% (v / v).

[0015] In some embodiments, the culture conditions are: a rotation speed of 160-180 rpm, 25-30° C., and a culture time of 12-15 h; in some specific embodiments, the culture conditions are: a rotation speed of 180 rpm, 30° C., and a culture time of 12 h.

[0016] In some specific embodiments, in step (2), the agar-containing liquid culture medium comprises the following components in percentage by weight:

[0017] 0.12% agar, 3% NaCl, 0.5% (NH4)2SO4, 0.26% K2HPO4·3H2O, 0.1% MgSO4·7H2O, 0.01% FeSO4·7H2O, pH 7.5.

[0018] In some embodiments, the inoculation amount of the seed solution in step (2) is 2-2.5% (v / v); in some specific embodiments, the inoculation amount can specifically be 2.5% (v / v).

[0019] In some embodiments, the culture conditions in step (2) are: a rotation speed of 160-180 rpm, 25-30° C., and a culture time of 50-60 h; in some specific embodiments, the culture conditions are: a rotation speed of 180 rpm, 30° C., and a culture time of 50 h.

[0020] The present invention also provides agar oligosaccharide prepared by the method described in the present invention.

[0021] The present invention studies the antioxidant properties of agar oligosaccharides (i.e., crude agar oligosaccharides with a molecular weight of less than 3 kDa) prepared by the method described in the present invention, and finds that the DPPH scavenging rate thereof at pH 6.5 to 7.5 is greater than 80%, and the DPPH scavenging rate at a temperature of 25 to 30° C. is greater than 85%, and has a significant antioxidant effect.

[0022] Based on the above effects, the present invention also provides the use of the agar oligosaccharide in the preparation of antioxidant products.

[0023] The present invention isolates a strain of Alteromonas macleodii QZ9-9 from the large seaweed Gracilaria, which was deposited in the China Center for Type Culture Collection on March 23, 2022, with a deposit number of CGMCCNO.24568. The strain can fully degrade agar into agar oligosaccharides with a molecular weight of less than 3kDa, and the obtained agar oligosaccharides have significant antioxidant effects.

[0024] Biological Deposit Description

[0025] QZ9-9, classification name: Alteromonas macleodii, was deposited on March 23, 2022 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No.24568. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the strain morphology;

[0027] Figure 2 This is the Gram staining picture of the strain;

[0028] Figure 3 The scanning electron micrograph of the strain;

[0029] Figure 4 The hydrolysis circle changes of agar degradation by strains in different time periods;

[0030] Figure 5 This is the phylogenetic tree of strain QZ9-9;

[0031] Figure 6 The antioxidant activity of agar oligosaccharides less than 3 kDa;

[0032] Figure 7 The effect of temperature on the antioxidant activity of agar oligosaccharide;

[0033] Figure 8 The effect of pH on the antioxidant activity of agar oligosaccharide;

[0034] Fig. 9 Comparative results of the antioxidant properties of agar oligosaccharides prepared by strain QZ9-9 and strain QZ8-6. DETAILED DESCRIPTION

[0035] The present invention discloses a strain of Alteromonas mesquite and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0036] The present invention will be further described below in conjunction with embodiments:

[0037] Example 1 Screening and Identification of Alteromonas macleodii QZ9-9

[0038] 1.1 Culture medium

[0039] (1) The mass volume percentage of each component in the agar liquid culture medium is: 0.12% agar, 3% NaCl, 0.5% (NH4)2SO4, 0.26% K2HPO4·3H2O, 0.1% MgSO4·7H2O, 0.01% FeSO4·7H2O, pH 7.5.

[0040] (2) The mass volume percentage of each component in the agar solid culture medium is: 1.5% agar, 3% NaCl, 0.5% (NH4)2SO4, 0.26% K2HPO4·3H2O, 0.1% MgSO4·7H2O, 0.01% FeSO4·7H2O, pH 7.5.

[0041] 1.2 Strain screening

[0042] 1.2.1 Sample collection: Collect large seaweed Gracilaria off the coast of Hainan, take 5 g of sample, add 50 mL PBS buffer, grind in a sterile grinder and pour into a 100 mL EP tube, shake it up and down gently, let it stand for 10 min and then set aside.

[0043] 1.2.2 Initial screening of strain QZ9-9: 10 μL of treated Gracilaria sample was inoculated into liquid culture medium with agar as the sole carbon source for fermentation culture at 30°C and 180 rpm for 24 h, and subcultured three times.

[0044] 1.2.3 Rescreening of strain QZ9-9: 3 μL of the fermentation liquid after subculture was applied to agar solid medium, and after colonies grew, streaking was performed several times until a single colony was isolated ( Figure 1 ).

[0045] 1.3 Morphological characteristics of strain QZ9-9

[0046] Strain QZ9-9 is a Gram-negative bacillus. Its colonies are moist, light yellow, round, with neat edges. It is an aerobic bacterium that can degrade agar and has a hydrolysis zone on solid culture medium ( Figure 1-3 ), and the diameter of the hydrolysis zone continued to expand with time (5% Lugol's iodine solution staining) ( Figure 4 ).

[0047] 1.4 Molecular biological identification of strain QZ9-9

[0048] After the genome of QZ9-9 was extracted, 16S rDNA sequencing and identification were performed, and the strain was identified as Alteromonas macleodii. We named it Alteromonas macleodii QZ9-9 ( Figure 5 ).

[0049] The 16S rRNA full gene sequence is 1381 bp long, and the sequence is shown in SEQ ID NO: 1.

[0050] 1.5 Method for preparing agar oligosaccharide by strain QZ9-9

[0051] (1) Alteromonas mesquite QZ9-9 was inoculated into 2216E seed culture medium at an inoculum size of 2.5% (v / v), the rotation speed was 180 rpm, the liquid volume was 25%, and the culture was carried out at 30° C. for 12 h to obtain the seed solution.

[0052] (2) Inoculate the seed liquid into agar liquid medium at an inoculum rate of 2.5% (the medium for preparing agar oligosaccharide is agar liquid medium: 0.12% agar, 3% NaCl, 0.5% (NH4)2SO4, 0.26% K2HPO4·3H2O, 0.1% MgSO4·7H2O, 0.01% FeSO4·7H2O, pH 7.5). Cultivate at 180 rpm, 30°C for 50 h to obtain fermentation liquid, remove the bacteria by centrifugation, and obtain supernatant;

[0053] (3) The supernatant is subjected to molecular interception method to obtain crude agar oligosaccharides with a molecular weight less than 3 kDa using an Ultra centrifuge tube.

[0054] 1.6 Antioxidant characteristics of QZ9-9 agar oligosaccharide

[0055] 1.6.1 Antioxidant activity of agar oligosaccharides less than 3 kDa

[0056] The antioxidant properties of agar oligosaccharides were evaluated using the DPPH free radical scavenging method. A 0.1 mM DPPH solution was prepared using anhydrous ethanol. First, the antioxidant properties of agar oligosaccharides <3 kDa were investigated. 50 μL of DPPH solution was mixed with 150 μL of agar oligosaccharide solution <3 kDa (pH 7.0). After incubation at 25°C in the dark for 60 minutes, the absorbance of the reaction solution was measured at 517 nm. Anhydrous ethanol was used instead of the sample as a negative control to calculate the antioxidant efficiency of agar oligosaccharides less than 3 kDa. The results showed that the DPPH scavenging rate of agar oligosaccharides <3 kDa after molecular entrapment was 88%, which was nearly 68% higher than the same volume of fermentation broth in step (2) of section 1.5, showing strong antioxidant properties ( Figure 6 ).

[0057] 1.6.2 Effect of temperature on the antioxidant activity of agar oligosaccharide

[0058] 50 μL DPPH solution was mixed with 150 μL agar oligosaccharide solution of <3 kDa (pH 7.0), and incubated at different temperatures in the dark for 60 min. The absorbance of the reaction solution was measured at 517 nm, and anhydrous ethanol was used instead of the sample as a negative control to investigate the antioxidant efficiency of <3 kDa agar oligosaccharide at different temperatures. The results showed that the DPPH scavenging rate was the best in the temperature range of 25-30°C and was >87% ( Figure 7 ).

[0059] 1.6.3 Effect of pH on the antioxidant activity of agar oligosaccharide

[0060] First, the pH of the <3kDa agar oligosaccharide solution was adjusted to 5 to 11. Then, 50μL of DPPH solution was mixed with 150μL of <3kDa agar oligosaccharide solution, and the absorbance of the reaction solution was measured at 517nm after incubation at 25°C in the dark for 60min. Anhydrous ethanol was used instead of the sample as a negative control to investigate the antioxidant efficiency of <3kDa agar oligosaccharide at different temperatures. The results showed that the DPPH clearance rate was best in the pH range of 6.5 to 7.5 and was >83%.

[0061] Comparative Example 1

[0062] In the strain screening of Example 1, the present invention also obtained strain QZ8-6, and the strain QZ8-6 was used to ferment and prepare agar oligosaccharides according to the method in Section 1.5, and the antioxidant effect of the obtained agar oligosaccharides was tested according to the method in Section 1.6.1. The results are shown in Fig. 9 .

[0063] The results showed that the DPPH clearance rate of agar oligosaccharides with a molecular weight less than 3 kDa prepared by strain QZ8-6 degrading agar was much lower than that of the strain QZ9-9 of the present invention, which was about 14%, and the antioxidant effect was far inferior to that of the strain of the present invention.

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Alteromonas macleodii with a deposit number of CGMCC NO.24568.

2. Use of the Alteromonas mesquite described in claim 1 in producing agarase and preparing agar-oligosaccharides.

3. A method for preparing agar oligosaccharide, It is characterized in that The invention is prepared by degrading agar by the Alteromonas mesquite described in claim 1.

4. The method according to claim 3, It is characterized in that include: Step (1): inoculating the Alteromonas mackeri with a deposit number of CGMCC NO.24568 into a seed culture medium, culturing, and obtaining a seed solution; Step (2): inoculating the seed liquid into a liquid culture medium containing agar, culturing, and removing the bacterial cells by centrifugation to obtain a supernatant; Step (3): The supernatant is subjected to molecular interception and centrifugation to collect components with a molecular weight less than 3 kDa.

5. The method according to claim 4, It is characterized in that In step (1), the inoculation amount of the Alteromonas mesquite is 2-2.5% (v / v); the culture conditions are: a rotation speed of 160-180 rpm, 25-30° C., and a culture time of 12-15 hours.

6. The method according to claim 4, It is characterized in that In step (2), the mass volume percentage of each component in the liquid culture medium containing agar is: 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.

7. The method according to claim 4, It is characterized in that In step (2), the inoculation amount of the seed solution is 2.5% (v / v); the culture conditions are: a rotation speed of 160-180 rpm, 25-30° C., and a culture time of 50-60 h.

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