A strain of Vibrio natriuresis and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of Vibrio natriegens and its application. The present invention isolates a strain of Vibrio natriegens V-Z9 from the large seaweed Gracilaria salsa, which was deposited in the China Center for Type Culture Collection on March 23, 2022, with a deposit number of CGMCC No. 24570. Compared with the existing Vibrio natriegens, the strain can fully degrade λ-carrageenan into λ-carrageenan oligosaccharides with a molecular weight of 10 to 30 kDa. The obtained λ-carrageenan oligosaccharides have a significant antioxidant effect.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, in particular to a strain of Vibrio natriuresis and application thereof. Background Art

[0002] Large seaweeds are valuable resources for developing marine polysaccharide functional foods and identifying novel polysaccharide drug lead compounds. They contain a diverse array of λ-carrageenan polysaccharides with diverse structures. However, the current supply of λ-carrageenan polysaccharides is significantly insufficient, and production methods are underdeveloped. Furthermore, λ-carrageenan polysaccharides suffer from large molecular weight, low water solubility, and poor absorption. Consequently, λ-carrageenan polysaccharides are sold only as raw materials abroad or extracted using simple, extensive processing techniques for use as food or food additives.

[0003] Compared with λ-carrageenan polysaccharides, λ-carrageenan oligosaccharides are not only rich in sources, diverse in types, stable in properties, widely used, and unique in activity, 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, such as antiviral, antioxidant, anticoagulant, and immune regulation.

[0004] Common methods for preparing λ-carrageenan oligosaccharides mainly include chemical methods and physical methods, but existing methods have disadvantages such as destroying the structure of polysaccharides and producing harmful by-products. Summary of the Invention

[0005] The present invention provides a strain of Vibrio natriuresis and an application thereof, which are used to overcome at least one technical problem existing in the prior art.

[0006] In view of this, the present invention provides a strain of Vibrio natriuresis and its application, which can degrade λ-carrageenan into λ-carrageenan oligosaccharides with a degradation rate greater than 85%. The obtained λ-carrageenan oligosaccharides have significant antioxidant effects.

[0007] The present invention isolated strain V-Z9 from the large seaweed Gracilaria spp., a gram-negative bacillus with moist, pale yellow, round colonies and neat edges. It is an aerobic bacterium that can degrade λ-carrageenan and produces hydrolysis zones on solid culture media. It also produces distinct clear zones after staining with Lu's iodine solution. The nucleotide sequence of the 16S rRNA gene of the strain is shown in SEQ ID NO: 1. Morphological analysis and the 16S rRNA gene analysis identified the strain as Vibrio natriegens, with a deposit number of CGMCC No. 24570.

[0008] The present invention also provides the use of the Vibrio natriureticus in the preparation of λ-carrageenan oligosaccharides.

[0009] The present invention also provides a method for preparing λ-carrageenan oligosaccharide, which is prepared by degrading λ-carrageenan with the Vibrio natriureticus V-Z9 of the present invention.

[0010] In some embodiments, the method for preparing λ-carrageenan oligosaccharides provided by the present invention specifically comprises:

[0011] Step (1): inoculating Vibrio natrii with a deposit number of CGMCC No. 24570 into a seed culture medium, culturing, and obtaining a seed solution;

[0012] Step (2): inoculating the seed liquid into a liquid culture medium containing λ-carrageenan, 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 of 10 to 30 kDa.

[0014] In some embodiments, the inoculation amount of Vibrio natriuresis V-Z9 in step (1) is 2-3% (v / v); in some specific embodiments, the inoculation amount can be specifically 3% (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 15 h.

[0016] In some specific embodiments, in step (2), the liquid culture medium containing λ-carrageenan includes the following components in mass volume percentages:

[0017] 0.15% λ-carrageenan, 2.5% NaCl, 0.35% (NH4)2SO4, 0.1% K2HPO4·3H2O, 0.2% MgSO4·7H2O, 0.02% FeSO4·7H2O, pH 7.

[0018] In some embodiments, the inoculation amount of the seed solution in step (2) is 1-3% (v / v); in some specific embodiments, the inoculation amount can be specifically 3% (v / v).

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

[0020] The present invention also provides λ-carrageenan oligosaccharide prepared by the method of the present invention.

[0021] The present invention studies the antioxidant properties of λ-carrageenan oligosaccharides (i.e., crude λ-carrageenan oligosaccharides with a molecular weight of 10 to 30 kDa) prepared by the method described in the present invention, and finds that the DPPH scavenging rate thereof is greater than 85% at a pH of 6.5 to 7.5 and at a temperature of 25 to 30°C, and has a significant antioxidant effect.

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

[0023] The present invention isolated a strain of Vibrio natriegens V-Z9 from the large seaweed Gracilaria salsa and deposited it with the China Center for Type Culture Collection on March 23, 2022, under the accession number CGMCC No. 24570. This strain is capable of fully degrading λ-carrageenan into λ-carrageenan oligosaccharides with a molecular weight of 10 to 30 kDa. The resulting λ-carrageenan oligosaccharides have significant antioxidant properties.

[0024] Biological Deposit Description

[0025] V-Z9, classification name: Vibrio natriegens, 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. 24570. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 is the strain morphology;

[0028] Figure 2 is the scanning electron micrograph of the strain;

[0029] Figure 3 This is the hydrolysis cycle (Lu's iodine solution) diagram of the strain degrading λ-carrageenan;

[0030] Figure 4 This is the phylogenetic tree of strain V-Z9;

[0031] Figure 5 The antioxidant activity of λ-carrageenan oligosaccharides of 10 to 30 kDa;

[0032] Figure 6 is the effect of temperature on the antioxidant activity of λ-carrageenan oligosaccharides;

[0033] Figure 7 The effect of pH on the antioxidant activity of λ-carrageenan oligosaccharides;

[0034] Figure 8 The antioxidant activity of 10-30 kDa λ-carrageenan oligosaccharides in the fermentation broth at different time periods. DETAILED DESCRIPTION

[0035] The present invention discloses a strain of Vibrio natrii 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 noted that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can 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 the embodiments:

[0037] Example 1 Screening and Identification of Vibrio natriegens V-Z9

[0038] 1.1 Culture medium

[0039] (1) The mass volume percentage of each component in the λ-carrageenan liquid culture medium is: 0.15% λ-carrageenan, 2.5% NaCl, 0.35% (NH4)2SO4, 0.1% K2HPO4·3H2O, 0.2% MgSO4·7H2O, 0.02% FeSO4·7H2O, and the pH is 7.

[0040] (2) The mass volume percentage of each component in the λ-carrageenan solid culture medium is: 1.2% agar, 2% λ-carrageenan, 2.5% NaCl, 0.35% (NH4)2SO4, 0.1% K2HPO4·3H2O, 0.2% MgSO4·7H2O, 0.02% FeSO4·7H2O, and the pH is 7.

[0041] 1.2 Strain screening

[0042] 1.2.1 Sample collection: Gracilaria off the coast of Hainan was collected. 5 g of sample was added to 50 mL of PBS buffer, ground in a sterile grinder, and poured into a 100 mL EP tube. The sample was gently shaken up and down and allowed to stand for 10 min before use.

[0043] 1.2.2 Initial screening of strain V-Z9: 10 μL of treated Gracilaria sample was inoculated into liquid culture medium with λ-carrageenan as the sole carbon source for fermentation. The culture was cultured at 30°C and 180 rpm for 48 h and passaged three times.

[0044] 1.2.3 Rescreening of strain V-Z9: 3 μL of the fermentation broth after subculture was applied to a λ-carrageenan solid medium. After the colonies grew, streak the culture medium several times until a single colony was isolated ( Figure 1 ).

[0045] 1.3 Morphological characteristics of strain V-Z9

[0046] Strain V-Z9 is a Gram-negative bacillus. Its colonies are moist, light yellow, round, with neat edges. It is an aerobic bacterium that can degrade λ-carrageenan and has a hydrolysis zone on solid culture medium. Figure 1-2 ) and after staining with Lugol's iodine solution, there is an obvious transparent circle (5% Lugol's iodine solution staining) ( Figure 3 ).

[0047] 1.4 Molecular biological identification of strain V-Z9

[0048] After the genome of V-Z9 was extracted and 16S rDNA sequencing and identification were performed, the strain was identified as Vibrio natriegens, and we named it Vibrio natriegens V-Z9 ( Figure 4 ).

[0049] The full 16S rRNA gene sequence is 1374 bp long, and the sequence is shown in SEQ ID NO: 1.

[0050] 1.5 Method for preparing λ-carrageenan oligosaccharides by strain V-Z9

[0051] (1) Vibrio natriuresis V-Z9 was inoculated into 2216E seed culture medium at a rate of 3% (v / v), the rotation speed was 180 rpm, the liquid volume was 30%, and the culture was carried out at 30° C. for 15 h to obtain the seed solution.

[0052] (2) The seed liquid was inoculated into a λ-carrageenan liquid culture medium at an inoculum size of 3% (the culture medium for preparing λ-carrageenan oligosaccharides is a λ-carrageenan liquid culture medium: 0.15% λ-carrageenan, 2.5% NaCl, 0.35% (NH4)2SO4, 0.1% K2HPO4·3H2O, 0.2% MgSO4·7H2O, 0.02% FeSO4·7H2O, pH 7). The culture was carried out at 180 rpm and 30°C for 60 h to obtain a fermentation broth, which was then centrifuged to remove the bacterial cells and obtain a supernatant.

[0053] (3) The supernatant was subjected to molecular interception method using an Ultra centrifuge tube to obtain crude λ-carrageenan oligosaccharides with a molecular weight of 10 to 30 kDa.

[0054] 1.6 Antioxidant properties of V-Z9λ-carrageenan oligosaccharides

[0055] 1.6.1 Antioxidant Activity of 10–30 kDa λ-Carrageenan Oligosaccharides

[0056] The antioxidant properties of λ-carrageenan oligosaccharides were evaluated using the DPPH free radical scavenging method. 0.1mM DPPH solution was prepared using anhydrous ethanol. First, the antioxidant properties of 10-30kDa λ-carrageenan oligosaccharides were investigated. 50μL DPPH solution was mixed with 150μL 10-30kDa λ-carrageenan oligosaccharide solution (pH 7.0). After incubation at 25°C in the dark for 60min, the absorbance of the reaction solution was measured at 517nm. Anhydrous ethanol was used instead of the sample as a negative control to calculate the antioxidant efficiency of 10-30kDa λ-carrageenan oligosaccharides. The results showed that the DPPH scavenging rate of 10-30kDa λ-carrageenan oligosaccharides after molecular entrapment was 85%, which was nearly 57% higher than that of the same volume of fermentation broth in step (2) of section 1.5, showing strong antioxidant properties ( Figure 5 ).

[0057] 1.6.2 Effect of temperature on the antioxidant activity of λ-carrageenan oligosaccharides

[0058] 50 μL of DPPH solution was mixed with 150 μL of 10-30 kDa λ-carrageenan oligosaccharide solution (pH 7.0). After incubation at different temperatures in the dark for 60 min, the absorbance of the reaction solution was measured at 517 nm. Anhydrous ethanol was used instead of the sample as a negative control to investigate the antioxidant efficiency of 10-30 kDa λ-carrageenan oligosaccharide at different temperatures. The results showed that the DPPH scavenging rate was the best in the temperature range of 25-35°C and was >85% ( Figure 6-7 ).

[0059] 1.6.3 Effect of pH on the antioxidant activity of λ-carrageenan oligosaccharides

[0060] First, the pH of a 10-30 kDa λ-carrageenan oligosaccharide solution was adjusted to 5-11. Subsequently, 50 μL of DPPH solution was mixed with 150 μL of a 10-30 kDa λ-carrageenan oligosaccharide solution. After incubation at 30°C in the dark for 60 minutes, the absorbance of the reaction solution was measured at 517 nm. Anhydrous ethanol was used as a negative control in place of the sample to investigate the antioxidant efficiency of the 10-30 kDa λ-carrageenan oligosaccharide at different temperatures. The results showed that the DPPH scavenging rate was optimal in the pH range of 6-7.5, with a consistently greater than 85% rate.

[0061] 1.6.4 Antioxidant Activity of 10–30 kDa λ-Carrageenan Oligosaccharides in Fermentation Broth at Different Time Periods

[0062] Fermentation broths at different fermentation stages were selected, centrifuged to remove the bacteria, and the supernatant was obtained. The supernatant was used by molecular interception to obtain crude λ-carrageenan oligosaccharides with a molecular weight of 10 to 30 kDa at different fermentation stages. 50 μL of DPPH solution was mixed with 150 μL of 10 to 30 kDa λ-carrageenan oligosaccharide solution at each time period (pH 7.0), incubated at 30°C in the dark for 60 minutes, and the absorbance of the reaction solution was measured at 517 nm. Anhydrous ethanol was used instead of the sample as a negative control to investigate the antioxidant efficiency of 10 to 30 kDa λ-carrageenan oligosaccharides in fermentation broths at different time periods. The results showed that the DPPH clearance rate of 10 to 30 kDa λ-carrageenan oligosaccharides in the fermentation broth after 60 hours of fermentation reached an optimal level of about 85%, and then entered a plateau period (60 to 90 hours). After the plateau period, the DPPH clearance rate of 10 to 30 kDa λ-carrageenan oligosaccharides in the fermentation broth gradually decreased ( Figure 8 ).

[0063] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Vibrio natriegens with a deposit number of CGMCC No. 24570.

2. Vibrio natriuresis according to claim 1, characterized in that The nucleotide sequence of its 16S rRNA gene is shown in SEQ ID NO:

1.

3. Use of the Vibrio natriureticus according to claim 1 or 2 in the preparation of λ-carrageenan oligosaccharides.

4. A method for preparing λ-carrageenan oligosaccharides, characterized in that: The invention is prepared by degrading λ-carrageenan by the Vibrio natriureticus according to claim 1 or 2.

5. The method according to claim 4, characterized in that include: Step (1): inoculating Vibrio natrii with a deposit number of CGMCC No. 24570 into a seed culture medium, culturing, and obtaining a seed solution; Step (2): inoculating the seed liquid into a liquid culture medium containing λ-carrageenan, culturing, and removing the bacteria by centrifugation to obtain a supernatant; Step (3): subjecting the supernatant to molecular interception and centrifugation to collect components with a molecular weight of 10 to 30 kDa.

6. The method according to claim 5, characterized in that In step (1), the inoculation amount of the Vibrio natrii is 2-3% (v / v); the culture conditions are: a rotation speed of 160-180 rpm, a temperature of 25-30° C., and a culture time of 12-15 hours.

7. The method according to claim 5, characterized in that In step (2), the mass volume percentage of each component in the liquid culture medium containing λ-carrageenan is: 0.15% λ-carrageenan, 2.5% NaCl, 0.35% (NH4)2SO4, 0.1% K2HPO4·3H2O, 0.2% MgSO4·7H 2 O, 0.02% FeSO4·7H2O, pH 7.

8. The method according to claim 5, characterized in that In step (2), the inoculation amount of the seed solution is 1-3% (v / v); the culture conditions are: a rotation speed of 160-180 rpm, a temperature of 25-30° C., and a culture time of 50-60 h.

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