AU2021441277A1 - Leuconostoc citreum strain and use thereof - Google Patents

Abstract

The present invention discloses a Leuconostoc citreum strain and use thereof. A classification name of the strain is Leuconostoc citreum, and a preservation number thereof is CGMCC NO.6431. A genome of the strain has two continuously arranged and normally expressed levansucrase encoding genes. The levansucrase encoding genes encode and express three levansucrases with different molecular weights. The molecular weights of the levansucrases are 130 kDa, 90 kDa and 80 kDa respectively. Three kinds of natural levansucrases with different molecular weights can be obtained by single fermentation of the Leuconostoc citreum provided by the present invention, thereby greatly improving the preparation efficiency of the levansucrases. 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 250kDa 150 kDa 100 kDa 75 kDa A B C FIG. 4

Description

Leuconostoc Citreum Strain and Use Thereof

TECHNICAL FIELD

The present invention relates to the technical field of microorganisms, and in

particular, to a Leuconostoc citreum strain and use thereof.

BACKGROUND

Levan is a class of fructose macromolecular polymers derived from plants or

microorganisms. The backbone of levan is composed of predominantly of P-(2,6) fructoside

bonds with occasionalp-(2,1) linkage in the branch chains. Studies have shown that

microbial-derived Levan has the anti-tumor, anti-diabetic, immune-enhancing, blood lipid

reducing and other functions. In addition, the Levan can also be used in the preparation of

nanomaterials and medicine carriers. In order to improve the purity of the product Levan, a

levansucrase is usually extracted from the microorganisms, and then reacts with a substrate to

obtain higher quality Levan. However, there are few microorganisms known to produce the

levansucrase, and a few of microorganisms are pathogenic. In addition, almost all

levansucrase-producing bacteria can only synthesize a levansucrase with one molecular weight.

Therefore, finding a strain with safe status and expressing alternative levansucrase for

preparing levan to meet the market has become an urgent problem to be solved in this field.

SUMMARY

Based on the above technical problems, the present invention provides a Leuconostoc

citreum strain and use thereof.

In order to realize the above objectives, the present invention provides the following

technical solutions.

The present invention provides a Leuconostoc citreum strain. A classification name of the

strain is Leuconostoc citreum, and a preservation number thereof is CGMCC NO.6431.

Further, a genome of the strain has two continuously arranged and normally expressed

levansucrase encoding genes.

Further, the levansucrase encoding genes express three levansucrases with different

molecular weights, i.e 130 kDa, 90 kDa and 80 kDa respectively.

The present invention further provides use of aLeuconostoc citreum strain in preparation

of levansucrases.

Further, three kinds of natural levansucrases with different molecular weights are

prepared by single fermentation of the strain.

Compared with the prior solution, the present invention has the following beneficial

effects.

The present invention discloses a strain of Leuconostoc citreum for the first time. The

genome of the strain has two continuously arranged levansucrase encoding genes that are

capable of being expressed normally and have low homology to known homologous genes.

The homology between the levansucrase encoding genes of the present application and the

known homologous genes is less than 30%. More importantly, the two levansucrase encoding

genes can encode and express three kinds of natural levansucrases with different molecular

weights, so that the three kinds of natural levansucrases with different molecular weights can

be obtained by single fermentation of the Leuconostoc citreum, thereby greatly improving the

preparation efficiency of the levansucrases. The above characteristics make the technical

solutions with significant technical advantages, so the present invention has a good application

prospect in the field of preparation of the levansucrases.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a distribution profile of genes 1291 and 1292 on a genome of CGMCC

NO.6431;

FIG. 2 is an SDS-PAGE gel profile of proteins 1291 and 1292;

FIG. 3 is a homology comparison profile of the genes 1291 and 1292;

FIG. 4 is an in-situ polymerization activity of levansucrases in the fermentation broth

collected by (NH4)2SO4 precipitation and isolated by SDS-PAGE; and

FIG. 5 is a nuclear magnetic resonance profile of polysaccharide samples.

DETAILED DESCRIPTION OF EMBODIMENTS

The technical solutions in the embodiments of the present invention will be clearly and

completely described below in combination with the accompanying drawings in the

embodiments of the present invention. Obviously, the described embodiments are only a part

of the embodiments of the present invention, rather than all the embodiments. Based on the

embodiments of the present invention, all other embodiments obtained by those of ordinary

skill in the art on the premise of not paying out creative labor, fall within the scope of the

protection of the present invention.

The present application provides a Leuconostoc citreum strain and use thereof.

Specifically, a preservation number of Leuconostoc citreum is CGMCC NO.6431. A classification name of the strain is Leuconostoc citreum. The strain was preserved in the China

General Microbiological Culture Collection Center (CGMCC) on August 13, 2012. The

preservation address is: No. 3 Courtyard 1 West Beichen Road, Chaoyang District, Beijing

100101, China.

Further, a genome of the Leuconostoc citreum has two continuously arranged and normally expressed levansucrase encoding genes.

Further, a homology between the levansucrase encoding genes and known homologous

genes is less than 30%.

Further, the levansucrase encoding genes can encode and express three levansucrases

with different molecular weights.

Further, the molecular weights of the levansucrases are 130 kDa, 90 kDa and 80 kDa

respectively.

In another specific implementation, the present invention provides use of the Leuconostoc

citreum in preparation of the levansucrases.

Further, three kinds of natural levansucrases with different molecular weights can be

prepared by single fermentation of the Leuconostoc citreum.

Compared with the prior solution, the above technical solutions disclose a strain of

Leuconostoc citreum for the first time. A genome of the strain has two continuously arranged

levansucrase encoding genes that are capable of being expressed normally and have low homology to known homologous genes. More importantly, the two levansucrase encoding genes can encode and express three kinds of natural levansucrases with different molecular weights, so that the three kinds of natural levansucrases with different molecular weights can be obtained by single fermentation of the Leuconostoc citreum, thereby greatly improving the preparation efficiency of the levansucrases. The above characteristics make the technical solutions have significant technical advantages, so the present invention has a good application prospect in the field of preparation of the levansucrases.

The above specific implementations are further described below through the

embodiments, but this does not limit the present invention to the scope of the described

embodiments. Experimental methods that do not specify specific conditions in the following

embodiments are selected according to conventional methods and conditions, or according to

product specifications.

In the following embodiments, all raw materials are commercially available and meet

relevant national standards.

Embodiment 1

A method for determining levansucrase encoding genes, specifically included the follows.

Whole genome sequencing was conducted on Leuconostoc citreum CGMCC NO.6431.

Two continuously arranged levansucrase encoding genes were found from a whole genome

sequence, named 1291 and 1292 respectively (see FIG. 1), and specific sequences thereof

were as follows.

The gene sequence 1291:

MNMKETTTRKKLYKSGKVWVAAATAFAVMGVSAVTTSISADTTASAVTAPTTAD KAAPAVTAPTTADKAAPAVTAPTTADKAAPAVTAPTTADKAAPAVTAPTTADKATPAVA APTTATSAQVTELTKNGLQLVSDAKIDNFDVNKLSSRQINSLNAAADKYYQNPNKTPN SNAVTYKNFDDLIKQLQEQPKNIAIPQFNSQNIKNLPAVTTESALTGKIEDLDIWDSWM VQDAQTQKVADVQGHQVMFALAGSTKEPADTHIYMLTTPYKATTINSWQMVGPVFG YNAVPWSQEWSGSATVNKDGSIQLFYTRVTWNDTIKQNMQRLSTINIVVRPTNSNGL AIENVNNDHIIFDGDGKYYQNIEQTVNSEGDNFTLRDPHVIEVDGQRYLSFETNTGTN NPEGYENVTDLSNYGGSLHYNVTKLLELVGNDAAFRTATLANGALGLLRLTQEQNNP TVTQIYDPLVTSNMVTDEIERANIVPLNGKYYLFTDTRLEKSSLGSNWNKSTASDIAM LGYVSDTLFGDYKPLNINGVVLVANKTSNDRTATYSYYAVPVDGYSDRLLITSYMSNR GMEAGNGLNATTAPSFIIQINADGTTAVEQTITTQNDWVDPYNAVDPSMYGFPGQAVN IKMAINSSFRTDGVFLNAPYGANVTNEHGLSITSEHVGNTTDYNGMSTQITRQYITSDN ITWYLASLNNRSVWIDSRAFTTVPFTPRDMTSFVNFSGRKDGIFTNAPYGMDNAKYV GNINNYQGQSFVIGGQYYDRGITWNLIQVNGQSVWVDNRSFATNFTHDTDKKVFVNT TSNLDGLFLNAPYRQPGYKLAGLAKNYNNQTVTVSQQYFDDQGTVWSQVVLGGQT VWVDNHALAQMQVRDTNQQLYVNSNGRNDGLFLNAPYRGQGSQLIGMTADYNGQ HVQVTKQGQDAYGAQWRLITLNNQQVWVDSRALSTTIMQAMNDDMYVNSSQRTD GLWLNAPYTMSGAKWAGDTRSANGRYVHISKAYSNEVGNTYYLTNLNGQSTWIDKR AFTTTFDQVVALNATIVARQRPDGMFKTAPYGEAGAQFVDYVTNYSQQTVPVTKQHS DAQGNQWYLATVNGTQYWIDQRSFSPVVTKVVDYQAKIVPRTTRDGVFSGAPYGEV NAKLVNMATAYQNQVVHATGEYTNASGITWSQFALSGQEDKLWIDKRALQA

The gene sequence 1292:

MKQQESITRKKLYRSGKSWVAAATAFAVMGVSAVTTSISADTTASAVTAPTTADK AAPAVTAPTTADKAAPAVTAPATADKAAPAVTAPTTADKAAPAVTAPTTADKAAPAVTA PTTADKAAPAVTALTAPNTARLVEKDLPANNEISGLTTFGNNLVCDAGLAKSDLIKNLS KDQIDAINQAATKYYDDPAKKPFSNAITYKDFDQLINQFNESPKELSVPKFNKENIKD MPSLTTKDAESNEVSALDMWDTWSVQDAKTKTVANVNGFQMMFGLAGAPTLGDTH MYMLYAKYGATHIEDWKMAGSVFGYDAVNSNQEWSGSAALNDDGSIQLFYTRVKW NSKLEANYQELWTANIDVSVIPDNEIQIKSINNDHSLFAGDGFYYEQLDQIRGTESQHG ENFALRDPNIIETDSGRYLTFEAGTGQYRPSGKQNITDLSIYGGDLSYNVKAMLNTVSN SDWKSLAGRSNAALGLLKLSGNQSDPDVEKLYTPRITSILTSDEIERANIVPLNGKYYL FAAARFDRSFLGHSPKLQPGYNVMMLGYVSDKIDGDYRPLNGNGTVLVSNIDFNDRT ATYAYYPVAVDGYSDRLLVTGYMSNRGQKTNTGYNATTAPSFIIQINADGTTAVEQTIT TQNDWVDPYNAVDPSMYGFPGQAVNIKMAINSSFRTDGVFLNAPYGANVTNEHGLSI TSEHVGNTTDYNGMSTQITRQYITSDNITWYLASLNNRSVWIDSRAFTTVPFTPRDMT SFVNFSGRKDGIFTNAPYGMDNAKYVGNINNYQGQSFVIGGQYYDRGITWNLIQVNG QSVWVDNRSFATNFTHDTDKKVFVNTTSNLDGLFLNAPYRQPGYKLAGLAKNYNNQ TVTVSQQYFDDQGTVWSQVVLGGQTVWVDNHALAQMQVRDTNQQLYVNSNGRND GLFLNAPYRGQGSQLIGMTADYNGQHVQVTKQGQDAYGAQWRLITLNNQQVWVDS RALSTTIMQAMNDDMYVNSSQRTDGLWLNAPYTMSGAKWAGDTRSANGRYVHISK AYSNEVGNTYYLTNLNGQSTWIDKRAFTTTFDQVVALNATIVARQRPDGMFKTAPYG EAGAQFVDYVTNYSQQTVPVTKQHSDAQGNQWYLATVNGTQYWIDQRSFSPVVTK VVDYQAKIVPRTTRDGVFSGAPYGEVNAKLVNMATAYQNQVVHATGEYTNASGITW SQFALSGQEDKLWIDKRALQA

In order to further prove whether the above levansucrase encoding genes 1291 and 1292

can be expressed normally, a heterologous expression experiment was designed.

1) Protein expression in Escherichiacoli Correct expression plasmid transformed competent E. coli BL21 (DE3) or Transrosetta

was identified. Three single colonies were picked to be inoculated to 5 mL LB culture

mediums (containing corresponding antibiotics) respectively, cultured overnight at 37°C, and

taken out. Overnight cultures were inoculated to 800 mL LB culture medium (containing

corresponding antibiotics) respectively, cultured for 5 hours at 37°C (OD600~0.5), transferred

to 25°C for cooling for 0.5 hour. IPTG was added to a final concentration of 0.2 mM.

Expression was conducted for 16 hours at 25°C. After the expression was completed, a

bacterial solution was taken out and cooled in ice, and centrifuged for 15 minutes at 5000 rpm

at 4°C. Bacterial cells were collected, and washed once with a bacterium lysing buffer solution.

The bacterium lysing buffer solution was discarded and the bacterial cells were cryopreserved

at -80°C.

2) Protein separation and purification

The bacterial cells cryopreserved at -80°C were taken out to be evenly resuspended into a

bacterium lysing buffer solution in a ratio of the bacterial cells to the solution of 1:5. Bacteria

were broken three times by an ultra-high pressure bacterium breaker (a bacterium breaking

pressure of 1,000 bar) to a translucent state. Then, the treated bacterial solution was

centrifuged for 0.5 hour at 18,000 rpm. The supernatant was loaded onto a pre-equilibrated

column with 5 mL or 1 mL of HisTrap FF by using a peristaltic pump. The column was rinsed

with the bacterium breaking buffer solution until no protein was eluted (a maximum flow rate is 0.5 mL/min per 1 mL of column volume), then washed with a washing buffer solution until no protein was eluted, and finally eluted with an eluting buffer solution to generate a target protein. The eluting buffer solution containing the target protein was desalted with a desalting column. A leacheate was collected to obtain a purified target protein.

The above target protein was subjected to SDS-PAGE electrophoresis and in-situ activity

detection, and a method thereof was as follows.

Samples to be tested were mixed with Native laoding buffer, loaded to 4% to 20%

SDS-PAGE gradient gel (Nanjing GenScript Biotech Corp., China), and electrophoresed for

1.5 hours at a voltage of 110V. Half of obtained protein gel was used for Coomassie brilliant

blue staining, and the other half was embathed twice with a NaAc solution (20 mM, pH 5.5),

and then placed in a NaAc solution (20 mM, pH 5.5) containing 5% sucrose and 0.3%o NaN3.

After reaction for 48 hours at 30°C, the number and positions of bands of in-situ reaction

products were observed in a dark room to confirm the number and molecular weights of

levansucrases synthesized by the Leuconostoc citreum.

Results were shown in FIG. 2. Protein bands expressed by the genes 1291 and 1292 were

found on the SDS PAGE gel, indicating that the two levansucrase encoding genes could be

normally expressed.

Conclusion: the genome of the Leuconostoc citreum CGMCC NO.6431 had two

continuously arranged and normally expressed levansucrase encoding genes (1291 and 1292).

Embodiment 2 Homology comparison of levansucrase encoding genes

Genes 1291 and 1292 were subjected to homology comparison with other known

homologous genes (CAD48195.1, AA014618.1, WP010237336, AAB97111.1, AC15886.1,

BAA04475, AAC36458.1, CBJ48143.1, CCM43846.1, CPR14579.1, EHD23269.1,

AAT81165.1, AAL9386.1, all sequences were from the NCBI website) by using mega7

software. Results were shown in FIG. 3. The homology between the genes 1291 and 1292 and

the known homologous genes was less than 30%, indicating that the two levansucrase

encoding genes had significant uniqueness.

Embodiment 3 Detection of levansucrases in fermentation broth

1. Materials and methods

(a) Preparation of seeds (fermentation strains): lyophilized powder of Leuconostoc

citreum CGMCC NO.6431 was resuspended with a small amount of sterile distilled water, and

a loop of the resuspended solution was streaked onto an M17 agar plate (Merck Co. Germany)

containing 2%(w/v) sucrose, aerobically cultured for 24 hours at 28°C and taken out. A single

colony was inoculated into sterile 20 mL M17 broth (Merck Co. Germany) containing 2%(w/v)

sucrose, cultured for 24 hours at 28°C at 180 rpm and taken out. The culture was centrifuged

for 10 minutes at 9,000 rpm, and the supernatant was discarded. The bacterial cells were

washed twice with sterile distilled water, and then suspended with an original culture volume of sterile distilled water to obtain seeds for fermentation. After detection, a bacterial

concentration of a seed liquid was 1x109 CFU/mL.

(b) Preparation of a tomato juice and sucrose culture medium: ripe tomatoes were washed,

peeled, squeezed by a juicer, and filtered with a 100-mesh gauze to extract juice. The juice

was boiled for 5 minutes and centrifuged for 10 minutes at 8,000 g. A supernatant was taken, and 15%(w/v) sucrose was added. After heating for dissolving of sucrose, the mixture was

cooled to room temperature, and the pH value of the mixture was adjusted to 6.5 with

food-grade alkali. The mixture was sterilized for 20 minutes at 121°C and then cooled to room

temperature to obtain the sterile tomato juice and sucrose culture medium.

(c) In-situ activity detection of levansucrases in the BD 1707 fermented broth: 50 mL of

the fermentation broth was taken and centrifuged for 30 minutes at 15,000 g. A supernatant

was taken, and ammonium sulfate was slowly added until a saturation reaches 60%.

Refrigerating was conducted overnight. Centrifuging was conducted for 30 minutes at 20,000

g at 4°C. The precipitate was redissolved into a small amount of distilled water, loaded into a

dialysis bag with a molecular weight cut-off of 1000 Da, and placed in a1%o CaCl2 solution

for low-temperature dialysis for 24 hours. During this period, the water is changed five times.

The retatent in the dialysis bag was lyophilized. 5 mg of the above lyophilized sample was

dissolved into 0.5 mL of PBS, then mixed with native laoding buffer, loaded to 4% to 20%

SDS-PAGE gradient gel (Nanjing GenScript Biotech Corp., China), and electrophoresed for

1.5 hours at a voltage of110V. Half of obtained protein gel was stained with Coomassie

brilliant blue. The other half was embathed twice with a NaAc solution (20 mM, pH 5.5), and

then placed in a NaAc solution (20 mM, pH 5.5) containing 5% sucrose and 0.3%o NaN3. After

reaction for 48 hours at 30°C, the number and positions of bands with in-situ polymerization

activity were observed in a dark room to confirm the number and molecular weights of the

levansucrases synthesized by the Leuconostoc citreum.

2. Detection of the levansucrases in the fermentation broth

The Leuconostoc citreum seeds were aseptically inoculated into a tomato juice culture

medium containing 15%(v/v) sucrose and with pH of 6.5 at a ratio of 3%(v/v), and cultured

for 96 hours at 30°C at 200 rpm to obtain the fermentation broth. In-situ activity of the levansucrases in the fermentation broth was detected. Results were shown in FIG. 4. Through

in-situ polymerization on SDS-PAGE gel, three obvious polysaccharide bands were found,

indicating that the fermentation broth contained three kinds of levansucrases with different

molecular weights, and the molecular weights thereof were 130 kDa, 90 kDa and 80 kDa

respectively.

Conclusion: Leuconostoc citreum CGMCC NO.6431 could encode and express three

levansucrases with different molecular weights, and the molecular weights thereof were 130

kDa, 90 kDa and 80 kDa respectively.

Effect Embodiment 1 Comparison of molecular weights of levansucrases

Through document retrieval, molecular weights of three kinds of levansucrases disclosed

by the present invention were compared with known reports, and results were shown in Table

1.

Table Comparison of molecular weights of levansucrases from different sources

Molecular weight of Data Strain levansucrase provenance

130 kDa The present Leuconostoc citreum CGMCC NO.6431 90 kDa invention 80 kDa

Bacillus 56 kDa [1]

Lactobacillus reuteri 121 84772 Da [2]

Lactobacillus sanfranciscensisTMW 1.392 90 kDa [3]

Bacillus lichenformis RN-01 56 kDa [4]

Data sources:

[1] Ben Ammar Y, Matsubara T, Ito K, et al. Characterization of a thermostable

levansucrase from Bacillus sp. TH4-2 capable of producing high molecular weight levan at

high temperature [J]. Journal of Biotechnology, 2002, 99(2): 111-119.

[2] van, Hijum, Aft S, et al. Biochemical and molecular characterization of a levansucrase

from Lactobacillus reuteri.[J]. Microbiology, 2004, 150: 621-630.

[3] Tieking M , Ehrmann M A , Vogel R F , et al. Molecular and functional

characterization of a levansucrase from the sourdough isolate Lactobacillus sanfranciscensis

TMW 1.392.[J]. Applied Microbiology and Biotechnology, 2005, 66(6):655-663.

[4] Sangmanee S , Nakapong S , Kuttiyawong K , et al. Production and Immobilization of

Levansucrase[J]. Chiang Mai Journal of Science, 2015, 42(1):44-51.

After comparison, it was found that a homology between levansucrase encoding genes of

the present application and known homologous genes was less than 30%. Besides,3

levansucrases with varied molecule weight could simultaneously be expressed by Leuconostoc

citreum in fermentation of tomato juice supplemented with 15%(w/v) , wherein the

levansucrase with the molecular weight of 130 kDa was the largest known levansucrase with

catalytic activity.

Effect Embodiment 2 Preparation of levansucrases

1. Materials and methods

(a) Separation of levansucrases: a crude levansucrase extract was dissolved into distilled

water and loaded to an AKTA protein purification system (GE Company, USA), wherein a

filler was Sephacryl S-300 High Resolution, a flow rate was 1 mL/min, a column volume was

120 mL, and a detector was a UV detector. The eluent with strong adsorbance at 280 nm was

collected partially, and lyophilized individually. 5 mg of individually lyophilized samples were

taken and added into a NaAc solution (20 mM, pH 5.5) containing 5% sucrose and 0.3%o

NaN3. After incubation for 48 hours at 30°C, the glucose released was determined by using a glucose detection kit (Biosino Bio-Technology and Science Incorporation, China), and if so, the sample was a levansucrase.

2 Preparation of the levansucrases

The fermentation broth prepared in Embodiment 3 was centrifuged for 10 minutes at

8,000 g. A supernatant was taken, and lyophilized to obtain a crude levansucrase extract. The

extract was separated to obtain three levansucrase samples.

Effect Embodiment 3 Activity verification of levansucrase samples

20 mg of the three levansucrase samples prepared in Effect Embodiment 2 were dissolved

into a NaAc solution (20 mM, pH 5.5) containing 5% sucrose and 0.3%o NaN3, respectively.

After reaction for 48 hours at 30°C, centrifuging was conducted for 10 minutes at 15,000 g. A

supernatant was taken, and 3 volumes of absolute ethyl alcohol was added. The mixture was

cold stored overnight and subsequently centrifuged for 10 minutes at 15,000 g. The precipitate

was collected, dissolved into water, and then lyophilized in vacuum to obtain three

polysaccharide samples. The three polysaccharide samples were completely dissolved into

heavy water at a concentration of 10 mg/mL, respectively, and subjected to nuclear magnetic

resonance (NMR) determination by applying a nuclear magnetic resonance spectrometer

(Avance III 400 MHz, Bruker Company, Germany). It was found that NMR profiles of the

three polysaccharide samples were consistent (as shown in FIG. 5), and chemical shifts of the

NMR-1H spectrum and the NMR-13C spectrum of all the polysaccharide samples were

consistent with those of a levan standard product.

Conclusion: all the three kinds of levansucrases with different molecular weights

expressed by the Leuconostoc citreum CGMCC NO.6431 had polymerization activity and

could be used to synthesize levan.

It will be apparent for those skilled in the art that the present invention is not limited to

the details of the above exemplary embodiments, and the present invention may be embodied

in other specific forms without departing from the spirit or essential characteristics of the

present invention. Therefore, the embodiments should be considered to be exemplary and

non-restrictive in all respects. The scope of the present invention is defined by the appended

claims rather than the above description, which are therefore intended to include all changes that fall within the meanings and scopes of equivalent elements of the claims within the present invention. Any reference signs in the claims shall not be construed as limiting the involved claims.

In addition, it should be noted that although this specification is described according to

implementations, not each implementation only includes an independent technical solution,

and this description mode in the specification is only for the sake of clarity, and those skilled

in the art should take the specification as a whole, the technical solutions in respective

embodiments can also be appropriately combined to form other implementations that can be

understood by those skilled in the art.

Claims (6)

1. A Leuconostoc citreum strain, wherein a classification name of the strain is

Leuconostoc citreum, and a preservation number thereof is CGMCC NO.6431.

2. The Leuconostoc citreum strain according to claim 1, wherein a genome of the strain

has two continuously arranged and normally expressed levansucrase encoding genes.

3. The Leuconostoc citreum strain according to claim 2, wherein the levansucrase

encoding genes express three levansucrases with different molecular weights.

4. The Leuconostoc citreum strain according to claim 3, wherein the molecular weights of

the levansucrases are 130 kDa, 90 kDa and 80 kDa respectively.

5. Use of the Leuconostoc citreum strain according to any of claims 1 to 4 in preparation

of the levansucrases.

6. The use of the Leuconostoc citreum strain in the preparation of the levansucrases

according to claim 5, wherein three kinds of natural levansucrases with different molecular

weights are prepared by single fermentation of the strain.