Photobacterium rosenbergii and use thereof in preparation of carrageenan oligosaccharide

Abstract

The present invention belongs to the technical field of bioengineering. Disclosed are Photobacterium rosenbergii and a use thereof in the preparation of a carrageenan oligosaccharide. The Photobacterium rosenbergii is specifically Photobacterium rosenbergii GDSX-4 with a 16S rDNA sequence as set forth in SEQ ID NO: 1. The Photobacterium rosenbergii GDSX-4 provided by the present invention can degrade κ-carrageenan to obtain a κ-carrageenan oligosaccharide. A crude enzyme liquid is prepared by means of the Photobacterium rosenbergii GDSX-4, and a reaction is carried out in a substrate solution containing κ-carrageenan at 40°C for 0.5-72 hours to prepare κ-neocarratetraose, κ-neocarrahexaose, and κ-neocarrabiose. The present invention provides a method for preparing a carrageenan oligosaccharide, wherein the reaction is mild and the product is uniform.

Description

A strain of Photobacterium louversii and its application in the preparation of carrageenan oligosaccharides Technical Field

[0001] The invention belongs to the technical field of bioengineering and relates to a Photobacterium luckerii strain and application thereof in preparing carrageenan oligosaccharides. Background Art

[0002] Carrageenan, also known as carrageenan and keratin, is an acidic polysaccharide extracted from red algae. It has a repeating α-1,4-D-galactopyranose-β-1,3-D-galactopyranose disaccharide backbone. The safety of carrageenan remains controversial. On the one hand, carrageenan is a large sulfated polysaccharide that is poorly digested and absorbed in the body. On the other hand, studies have shown that carrageenan can easily cause gastrointestinal inflammation and intestinal immune responses. Furthermore, studies have found that carrageenan exhibits various biological activities, including lipid-lowering, anticoagulant, antithrombotic, immunomodulatory, and connective tissue growth stimulation, showing promising application prospects in the biopharmaceutical field. However, its large molecular weight and poor solubility and absorbability limit its application.

[0003] Carrageenan oligosaccharides are degradation products of carrageenan. Compared to carrageenan, they possess lower molecular weight, higher solubility, easier absorption, and improved stability and safety. Furthermore, due to the fully exposed active groups on the molecular chain, their activity is significantly enhanced compared to carrageenan. Currently, carrageenan degradation methods include chemical degradation (such as oxidative degradation and acid degradation); physical degradation (such as irradiation and microwave degradation); and enzymatic degradation. However, chemical hydrolysis is often too intense, causing structural damage to the product during the hydrolysis process, significantly limiting its application. Enzymatic degradation, on the other hand, maximizes the protection of the active groups of the reaction substrate from damage during the degradation process. The degradation products are uniform, the reaction conditions are mild, and the products are highly active, making it a promising preparation method. Therefore, providing a strain capable of producing carrageenan-degrading enzymes is of great significance to enrich the microbial resources for enzymatic degradation of carrageenan oligosaccharides. Summary of the Invention

[0004] To provide a strain capable of degrading carrageenan to produce carrageenan oligosaccharides, the present invention provides a strain of Photobacterium rosenbergii and its use in producing carrageenan oligosaccharides. Specifically, Photobacterium rosenbergii GDSX-4 is capable of degrading κ-carrageenan to produce κ-neocalabiose, κ-neocalametose, and κ-neocalahexaose. This method provides a mild reaction and uniform product for the production of carrageenan oligosaccharides.

[0005] To achieve the technical purpose of the present invention, on the one hand, the present invention provides a strain of Photobacterium rosenbergii, specifically Photobacterium rosenbergii GDSX-4, which was deposited in the Guangdong Provincial Microbial Culture Collection Center (5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province) on October 13, 2023, with the deposit number: GDMCC 63878; Photobacterium rosenbergii produces an enzyme that degrades carrageenan.

[0006] The present invention obtained strain GDSX-4 from Gracilaria coronopifolia through enrichment culture, primary screening, and secondary screening. Morphological identification revealed that strain GDSX-4 is a Gram-negative, rod-shaped bacterium. Molecular identification confirmed that strain GDSX-4 is Photobacterium rosenbergii. The 16S rDNA sequence of Photobacterium rosenbergii GDSX-4 is shown in SEQ ID NO: 1.

[0007] Furthermore, the culture conditions of Photorhabditis leucoderma provided by the present invention are: enrichment medium at 30°C, 150 rpm, and shaking culture for 24 hours. The enrichment medium includes: 10 g / L beef extract, 20 g / L tryptone, 21.120 g / L NaCl, 0.5820 g / L KCl, 0.820 g / L CaCl2, 3.620 g / L MgCl2·6H2O, 0.0820 g / L NaHCO3, 2.62 g / L MgSO4·7H2O, and a pH of 7.3.

[0008] Furthermore, the Photobacterium lutherii provided by the present invention can degrade carrageenan, specifically κ-carrageenan, and its degradation products are κ-carrageenan oligosaccharides. κ-carrageenan oligosaccharides include κ-neocalabiose (DP2), κ-neocalabiose (DP4), and κ-neocalabiose (DP6).

[0009] In another aspect, the present invention claims the use of the aforementioned Photobacterium luckerii in the preparation of carrageenan oligosaccharides. Photobacterium luckerii is capable of degrading κ-carrageenan to obtain κ-carrageenan oligosaccharides, which include κ-neokarabiose, κ-neokaratetraose, and κ-neokarahexaose.

[0010] Furthermore, the present invention cultivates Photorhabdus lutherii GDSX-4 in a solid enzyme-producing medium at 30°C for 48 hours, resuspends the cells, ultrasonically disrupts them, and centrifuges the supernatant to obtain a crude enzyme solution. The crude enzyme solution is then added to a κ-carrageenan solution and degraded at 40°C for 0.5 to 72 hours to obtain κ-carrageenan oligosaccharides. Preferably, the degradation time is 1 to 48 hours. More preferably, the degradation time is 48 hours.

[0011] On the other hand, the present invention claims an enzyme for degrading carrageenan, wherein the enzyme is prepared by Photobacterium rosenbergii GDSX-4.

[0012] On the other hand, the present invention seeks to protect a microbial agent containing the above-mentioned Photobacterium rosenbergii GDSX-4, which is used to prepare carrageenan oligosaccharides, and the carrageenan oligosaccharides include κ-neocalabiose, κ-neocalabotetrose and κ-neocalahexaose.

[0013] The present invention also claims a method for preparing carrageenan oligosaccharides, which are prepared using Photobacterium rosenbergii GDSX-4, or using a carrageenan-degrading enzyme, or using a microbial agent. The carrageenan oligosaccharides are prepared at a temperature of 40°C for 0.5 to 72 hours. The carrageenan oligosaccharides include κ-neocalabiose, κ-neocalatraose, and κ-neocalahexaose.

[0014] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0015] The present invention provides a bacterial strain capable of producing uniform products for enzymatically degrading carrageenan to produce carrageenan oligosaccharides. The present invention provides a carrageenan-degrading bacterial strain, Photobacterium rosenbergii GDSX-4, which is capable of degrading κ-carrageenan to produce κ-carrageenan oligosaccharides, including κ-neocalabiose, κ-neocalametetrose, and κ-neocalahexaose.

[0016] Thin-layer chromatography analysis revealed that after a degradation reaction lasting more than 30 minutes, the system primarily produced κ-neocalabiose, κ-neocalabiose, and κ-neocalabiose, along with some oligosaccharide components with a degree of polymerization greater than six. The concentration of oligosaccharide spots increased with degradation time, particularly after 48 hours, when oligosaccharides with a degree of polymerization greater than six decreased significantly, likely due to degradation of these oligosaccharides into κ-neocalabiose.

[0017] The present invention found through liquid phase mass spectrometry analysis that before 45 minutes of degradation reaction, the product was mainly κ-neocalabiose, with obvious detection peaks, and a small amount of κ-neocalabiose and κ-neocalahexaose were generated. After 1 hour of degradation reaction, the response peaks of the three oligosaccharide components were clearly seen. As the degradation time increased, the production of the three oligosaccharide components gradually increased, especially after 48 hours of degradation, the product production reached a peak. After the degradation reaction was completed, the final products were κ-neocalabiose, κ-neocalabiose and κ-neocalabiose. Among the completely generated degradation products, 44% was κ-neocalabiose, 32% was κ-neocalabiose, and 24% was κ-neocalabiose. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 shows images of the plate culture and microscopic examination of Photobacterium luckerii GDSX-4. Figure 1a shows a streak plate culture of Photobacterium luckerii GDSX-4; Figure 1b shows a plate culture of Photobacterium luckerii GDSX-4 after rescreening with Lugol's iodine staining; Figure 1c shows the Gram staining results of Photobacterium luckerii GDSX-4; and Figure 1d shows a scanning electron micrograph of Photobacterium luckerii GDSX-4.

[0020] Figure 2 is a phylogenetic tree of strain GDSX-4.

[0021] Figure 3 shows the reaction results of reducing sugar and DNS color reagent in the reaction system with different degradation times. Ctrl is the sample with 0h degradation.

[0022] Figure 4 shows thin-layer chromatography analysis of the products of κ-carrageenan degradation by Photobacterium lutherii GDSX-4. Ctrl is the sample after degradation for 0 hours; 2 is the standard κ-neocalabiose; 4 is the standard κ-neocalabiose; 6 is the standard κ-neocalabiose.

[0023] Figure 5 shows the results of mass spectrometry detection of κ-neocalabiose, κ-neocalatraose, and κ-neocalahexaose. DP2 is κ-neocalabiose; DP4 is κ-neocalatraose; and DP6 is κ-neocalahexaose.

[0024] Figure 6 shows the total ion chromatogram of the products of κ-carrageenan degradation detected by mass spectrometry using Photorhabdus luckeri GDSX-4. DP2 is κ-neocalabiose; DP4 is κ-neocalanetetrose; and DP6 is κ-neocalahexaose.

[0025] Figure 7 shows the peak area statistics of the products of κ-carrageenan degradation by Photobacterium lutherii GDSX-4 detected by mass spectrometry. DP2 is κ-neocalabiose; DP4 is κ-neocalanetetrose; and DP6 is κ-neocalahexaose. DETAILED DESCRIPTION

[0026] The following examples illustrate the technical solutions of the present invention, but the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0027] Enrichment medium: beef extract 10 g / L, tryptone 20 g / L, NaCl 21.120 g / L, KCl 0.5820 g / L, CaCl2 0.820 g / L, MgCl2·6H2O 3.620 g / L, NaHCO3 0.0820 g / L, MgSO4·7H2O 2.62 g / L, pH 7.3.

[0028] Solid screening medium: carrageenan 12 g / L, NaCl 15 g / L, NaNO3 20 g / L, CaCl2 0.1 g / L, MgSO4·7H2O 0.5 g / L, FeSO4 0.004 g / L, K2HPO4 1 g / L.

[0029] Solid enzyme production culture medium: carrageenan 15g / L, peptone 5g / L, yeast powder 1g / L, dissolved in 1L of filtered sterilized aged sea water.

[0030] Gracilaria serrata was provided by Guangdong Ocean University.

[0031] κ-Carrageenan was purchased from Qingdao Haida Marine Oligosaccharide Technology Co., Ltd. Example 1

[0032] This example provides the screening and identification of Photorhabdus luckerii GDSX-4.

[0033] 1. Screening of strains

[0034] Gracilaria coronopifolia was homogenized and incubated in an enrichment medium at 30°C, 150 rpm, and shaker for 24 hours. 100 μL of the enrichment medium was spread onto a solid screening medium and incubated in an inverted manner at 30°C for 48 hours. Colony growth was observed, and colonies with distinct pits were selected and repeatedly streaked onto the plate until a single colony was obtained (Figure 1, a). A single colony was inoculated into a solid enzyme production medium and incubated at 30°C for 48 hours to obtain the primary screening strain. 5 μL of the primary screening strain was dripped onto a solid screening medium containing carrageenan as the sole carbon source. After inverted incubation at 30°C for 48 hours, the strain was stained with 5 mL of Lugol's iodine solution (Figure 1, b), and the diameter of the transparent zone was measured with a vernier caliper.

[0035] As shown in Figures 1a and 1b, strain GDSX-4 can grow on solid screening medium containing carrageenan. Staining with Lugol's iodine solution reveals a distinct hydrolysis zone with a diameter of 3.0 cm. Colony morphology reveals that individual colonies are round, with neat edges, a moist, shiny, milky white surface, moderate size, and easy to pick up.

[0036] 2. Morphological identification of strains

[0037] The screened strains were Gram-stained and their morphology was observed under a microscope (Figure 1, c). Gram-stained bacterial cells showed purple for Gram-positive bacteria and red for Gram-negative bacteria. Bacterial morphology was observed using a scanning electron microscope (Figure 1, d).

[0038] As can be seen from Figure 1c and Figure 1d, the bacteria appear red, are Gram-negative bacteria, and are rod-shaped.

[0039] 3. Molecular identification of strains

[0040] DNA was extracted from the obtained single colony pure culture using the Ezup column-type bacterial genomic DNA extraction kit (purchased from Sangon Biotech Shanghai Co., Ltd.). The obtained DNA was subjected to full-length 16S rRNA amplification. The specific amplification experimental conditions and primer sequences are as follows:

[0041] Universal primer sequences: 27F (5′-AGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT3′).

[0042] Amplification system: 25 μL reaction system, DNA template, 1 μL each primer, 2.5 μL 10× PCR Buffer, 0.5 μL dNTP Mix (10 mM), 0.2 μL Taq enzyme (5 U / L), and deionized water to 25 μL. Taq enzyme was purchased from MBI (Fermentas).

[0043] Amplification conditions included pre-denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 90 seconds, followed by 10 minutes at 72°C. The PCR product was purified and sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing. The resulting 16S rDNA sequence of strain GDSX-4 is shown in SEQ ID NO: 1.

[0044] The sequencing results were submitted to the EZbio database for blast alignment. Sequences with high homology were downloaded and multiple sequence alignment was performed using ClustalX. The eight closest model strains were selected, and a phylogenetic tree was constructed using MEGA, as shown in Figure 2.

[0045] As shown in Figure 2, the homology between strain GDSX-4 and Photobacterium rosenbergii is 99.93%. Based on 16S rDNA comparison, strain GDSX-4 was identified as Photobacterium rosenbergii and named Photobacterium rosenbergii GDSX-4. Example 2

[0046] This example provides the use of Photorhabdus luckerii GDSX-4 in the preparation of carrageenan oligosaccharides.

[0047] 1. Collection of crude enzyme solution of Photobacterium louversii GDSX-4

[0048] The strain was cultured in a solid enzyme-producing medium at 30°C for 48 h, resuspended in Tris-HCl buffer at pH 8.0, and ultrasonically disrupted for 20 min. The disrupted liquid was centrifuged at 4°C and 12,000 rpm / min for 15 min, and the supernatant was taken as the crude enzyme solution.

[0049] 2. Preparation of carrageenan oligosaccharides

[0050] A 0.5 wt% κ-carrageenan substrate solution was prepared in Tris-HCl buffer (pH 8.0), and 10% of the crude enzyme solution was added based on the total reaction volume. The reaction was carried out at 40°C for 0 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 6 h, 12 h, 24 h, 27 h, 30 h, 48 h, and 72 h, respectively. The reaction was terminated by heating and boiling for 5 min and then cooled to room temperature.

[0051] 3. Analysis of κ-carrageenan oligosaccharides by thin layer chromatography

[0052] A silica gel thin layer plate was activated in a drying oven at 100°C for 1 h. 3 μL of the carrageenan oligosaccharide sample obtained by the above reaction was spotted on the silica gel plate. The sample at 0 h of reaction was used as a reference (Ctrl), and the standard substances κ-neocalabiose (DP2), κ-neocalaotetraose (DP4), and κ-neocalahexaose (DP6) were used as controls. The plate was developed in a developing agent of n-butanol:ethanol:water (3:2:2) and then blown dry. The plate was then soaked in a color developer (2 g of diphenylamine, 2 mL of aniline, 10 mL of 85% phosphoric acid, 1 mL of concentrated hydrochloric acid, and 100 mL of acetone) and blown dry. The plate was heated at 110°C for 10 min for color development (Figure 3). The thin layer chromatography results are shown in Figure 4.

[0053] As shown in Figure 3, as the degradation time increases, the color of the reaction system gradually becomes darker, indicating that the content of reducing sugar in the reaction system gradually increases with time.

[0054] As shown in Figure 4, only a small amount of oligosaccharides are produced in the system before 30 minutes of degradation by Photobacterium lutherii GDSX-4. After 30 minutes, the system primarily produces κ-neocalabiose, κ-neocalabiose, and κ-neocalabiose, in addition to some oligosaccharides with a degree of polymerization greater than six. The concentration of oligosaccharide spots increases with degradation time, especially after 48 hours, when oligosaccharides with a degree of polymerization greater than six decrease significantly, likely due to degradation of these oligosaccharides into κ-neocalabiose.

[0055] 4. Analysis of degradation product structure by liquid chromatography-mass spectrometry (Q-TOF-MS)

[0056] The reaction solution after thin-layer chromatography analysis was diluted to 50 μg / mL, filtered through a 0.22 μm microporous filter, and analyzed on a TOF-MS system. The chromatographic conditions were: injection volume 5 μL; mobile phase: acetonitrile:1 mM formic acid (1:1); flow rate 0.2 mL / min; column temperature 35°C. Mass spectrometry was performed using an ESI point spray ionization source in negative ion mode; mass scan range: m / z 50–2000 Da.

[0057] First, the mixed standard of κ-neocalabiose, κ-neocalabiose, and κ-neocalabiose was separated and identified by primary mass spectrometry (Figure 5), and the changes in the amount of oligosaccharides produced between 0.5 and 72 hours of the degradation reaction of Photorhabdus lutherii GDSX-4 were further detected (Figures 6 and 7).

[0058] As can be seen from Figure 5, the peak with a mass-to-charge ratio of m / z 403.05 is the ion peak of a disaccharide carrying a sulfate group, which is the characteristic ion peak of κ-neocalamiose [An-G4S]-, the peak with a mass-to-charge ratio of m / z 394.05 is the characteristic ion peak of κ-neocalamiose [(An-G4S)2]-, and the peak with a mass-to-charge ratio of m / z 391.05 is the characteristic ion peak of κ-neocalamiose [(An-G4S)3]-.

[0059] Figures 6 and 7 show that before 45 minutes of degradation, the primary product is κ-neocalabiose (DP2), with a distinct detection peak. Small amounts of κ-neocalabiose (DP4) and κ-neocalahexaose (DP6) are also produced, but the response values ​​are extremely low, making it impossible to integrate the peak areas. After 1 hour of degradation, distinct peaks for the three oligosaccharide components are visible. The production of these components gradually increases with degradation time, reaching a peak after 48 hours. Further increases in degradation time do not result in further product production. Upon completion of the degradation reaction, the final products are κ-neocalabiose, κ-neocalabiose, and κ-neocalabiose. Of the fully generated degradation products, 44% is κ-neocalabiose, 32% is κ-neocalabiose, and 24% is κ-neocalabiose.

[0060] The embodiments described above are some of the embodiments of the present invention, rather than all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A Photobacterium louversii, characterized in that The Photobacterium rosenbergii is Photobacterium rosenbergii GDSX-4, which was deposited in Guangdong Microbiological Culture Collection Center on October 13, 2023, with the deposit number: GDMCC 63878; The Photobacterium luckerii produces enzymes that degrade carrageenan.

2. Photobacterium luckerii according to claim 1, characterized in that The 16S rDNA sequence of the Photobacterium luckerii is shown in SEQ ID NO:

1.

3. Photobacterium luckerii according to claim 1, characterized in that The carrageenan is κ-carrageenan; The degradation product of carrageenan is kappa-carrageenan oligosaccharide.

4. Photobacterium luckerii according to claim 3, characterized in that The κ-carrageenan oligosaccharides include κ-neocalabiose, κ-neocalaotetraose and κ-neocalahexaose.

5. Use of the Photobacterium luckerii described in any one of claims 1 to 4 in the preparation of carrageenan oligosaccharides.

6. The use according to claim 5, characterized in that: The Photobacterium luckerii degrades κ-carrageenan to obtain κ-carrageenan oligosaccharides; The carrageenan oligosaccharides include κ-neocalabiose, κ-neocalatetraose and κ-neocalahexaose.

7. The use according to claim 6, characterized in that: The degradation temperature is 40° C., and the degradation time is 0.5 to 72 hours.

8. An enzyme for degrading carrageenan, characterized in that: The enzyme is prepared by the Photorhabdus luckerii described in claim 1.

9. A microbial agent, characterized in that: The microbial agent contains the Photobacterium luckerii described in claim 1; The Photobacterium luckerii is used for the preparation of carrageenan oligosaccharides; The carrageenan oligosaccharides include κ-neocalabiose, κ-neocalatetraose and κ-neocalahexaose.

10. A method for preparing carrageenan oligosaccharides, characterized in that: Using the Luteobacterium luckeri described in any one of claims 1 to 4 to prepare carrageenan oligosaccharides, or using the enzyme described in claim 8 to prepare carrageenan oligosaccharides, or using the microbial agent described in claim 9 to prepare carrageenan oligosaccharides; The temperature for preparing carrageenan oligosaccharides is 40° C., and the time for preparing carrageenan oligosaccharides is 0.5 to 72 hours; The carrageenan oligosaccharides include κ-neocalabiose, κ-neocalatetraose and κ-neocalahexaose.

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