KR20110135728A - Novel lactobacillus spp. strains for producing sweet-tasting protein, brazzein and a method for producing the brazzein using the same - Google Patents

Abstract

PURPOSE: A Lactobacillus sp. which produces sweet-tasting protein, Brazzein, is provided to highly produce Brazzein. CONSTITUTION: A Lactobacillus reuteri which expresses Brazzein has an amino acid sequence of sequence number 1. A method for preparing Brazzein comprises: a step of culturing Lactobacillus reuteri(KCTC18192P) strain in a medium containing 3-15 ug/ml of erythromycin and 10-50 ng/ml of nisin; and a step of isolating Brazzein form the culture liquid. The nisin is treated for three to four hours.

Description

Novel Lactobacillus spp.strains for producing sweet-tasting protein, Brazein and a method for producing the Brazzein using the same}

The present invention relates to a strain of Lactobacillus spp. That produces sweet protein brazain and a method for producing brazain using the same.

Sugar intake is associated with problems such as obesity, tooth decay and diabetes. These problems have led to reduced sugar intake and increased sugar intake. Artificial sweeteners include saccharin, cyclamate, aspartame, sucralose and stevioside, which are currently the most popular sweet substitutes that are much sweeter than sugar. to be. However, due to the finding that many artificial sweeteners are associated with bladder cancer, brain tumors and mental illness (National Cancer Institute Fact Sheet. 2006; Soffritti et al. 2005), new alternative sweeteners that are safe and beneficial to health There was a need for it.

Sweet protein is isolated from various African and South Asian fruits and has been used as a natural sweetener because it is about 500 to 4000 times more sweet by weight than sucrose (Faus 2000). Brazein is the smallest sweet plant protein (6,473 Da) and is composed of 54 amino acids. It is a Western African plant, Pentai Plandra, Brazeana and Pentadiplandra. It is isolated from brazzeana Baillon (Ming and Hellekant 1994), 500 times sweeter than sucrose by weight, and more stable against heat and pH due to disulfide bonds in the molecule. For this reason, as a potent sugar substitute, studies on brazain are being actively conducted (Assadi-Porter et al. 2000; Berlec et al . 2006; Ming and Hellekant 1994).

There are reports of expression of recombinant brazain in E. coli (Assadi-Porter et al. 2000), maize (Lamphear et al. 2005) and Lactococcus lactis (Berlec et al. 2006). To express brazain, a nisin-controlled expression system (NICE) was applied in L. lactis (de Ruyter et al. 1996.) In addition, optimization of fermentation conditions and plasmid / strains It was reported that the expression of brazain protein was increased by the change of the combination (Berlec et al. 2008, Berlec et al. 2009), but the expression level of brazain protein was still relatively low.

Bacteria belonging to the genus Lactobacillus spp. Have recently received a lot of attention as probiotics due to their putative health-conferring properties (Sanders 1999). Many studies have been preceded by a number of species, and complete genome sequences and already established genetic techniques are available (Kleerebezem et al. 2003; Wei et al. 1995). In view of the industrial and medical importance of the well-known Lactobacilli, it is of particular interest to understand the characteristics of lactobisilli (Wei et al., 1995).

Accordingly, the present inventors transformed the plasmid expressing the brazain into Lactobacillus reuteri, and then treated the nisin to produce a new strain expressing the brazain for replacing the sweetener. When the expression of brazain increases, the increase in expression is not only related to the concentration and treatment time of nisin, but also in the Lactobacillus reuteri, which has the highest stability of the inserted plasmid in the genus Lactobacillus. The present invention was completed by confirming that phosphorus production was the highest.

Assadi-Porter F M, Aceti DJ, Cheng H, Markley JL (2000) Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin. Arch Biochem Biophys 376: 252-258. Berlec A, Jevnikar Z, Majhenic AC, Rogelj I, Strukelj B (2006) Expression of the sweet-tasting plant protein brazzein in Escherichia coli and Lactococcus lactis: a path toward sweet lactic acid bacteria. Appl Microbiol Biotechnol 73: 158-165. Berlec A, Tompa G, Slapar N, Fonovic UP, Rogelj I, Strukelj B (2008) Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis. Lett Appl Microbiol 46: 227-231. Berlec A, Strukelj B (2009) Large increase in brazzein expression achieved by chaning the plasmid / strain combination of the NICE system in Lactococcus lactis. Lett Appl Microbiol 48: 750-755. Bryan EM, Bae T, Kleerebezem M, Dunny GM (2000) Improved vectors for nisin-controlled-expression in Gram-positive bacteria. Plasmid 44: 183-190. Chang CE, Pavlova SI, Tao L, Kim EK, Kim SC, Yun HS, So JS (2002) Molecular identification of vaginal Lactobacillus spp. isolated from Korean women. J Microbiol Biotechnol 12: 312-317. Faus I (2000) Recent developments in the characterization and biotechnological production of sweet-tasting proteins. Appl Microbiol Biotechnol 53: 145-151. Kleerebezem M, Boekhorst J, van Kranenburg R et al (2003) Complete genome sequence of Lactobacillus plantarum WCFS1. Proc Natl Acad Sci USA 100: 1990-1995. Lamphear BJ, Barker DK, Brooks CA et al (2005) Expression of the sweet protein brazzein in maize for production of a new commercial sweetener. J Plant Biotechnol 3: 103-114. Lee YW, Im SH, Xin CF, So JS (2009) Determination of optimal electrotransformation conditions for various Lactobacillus spp. Korean J Biotechnol Bioeng 24: 182-188. Ming D, Hellekant G (1994) Brazzein, a new high-potency thermostable sweet protein from Pentadiplandra brazzeana B. FEBS Lett 355: 106-108. National Cancer Institute Fact Sheet (2006) Artificial sweeteners and cancer: questions and answers. http://www.cancer.gov/cancertopics/factsheet/Risk/artificial-sweeteners#ref1. Accessed 7 January 2010. Pavan S, Hols P, Delcour J, Geoffroy M, Grangette C, Kleerebezem M, Mercenier A (2000) Adaptation of the nisin-controlled expression in Lactobacillus plantarum: a tool to study in vivo biological effects. Appl Environ Microbiol 66: 4427-4432. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, New York. Sanders TA (1999) Food production and food safety. BMJ 318: 1689 ◎ 693. Soffritti M, Belpoggi F, Esposti DD, Lambertini L (2005) Aspartame induces lymphomas and leukaemias in rats. Eur J Oncol 10: 107-116. Wei M, Rush CM, Norman JM, Hafner LM, Epping RJ, Timms P (1995) An improved methods for the transformation of Lactobacillus strains using electroporation. J Microbiol Meth 21: 97-109.

An object of the present invention is to provide a Lactobacillus spp. Strain producing a sweet protein Brazain (Brazzein) and a method for producing brazain using the same.

In order to achieve the above object, the present invention provides a Lactobacillus reuteri strain expressing the brazain deposited with accession number KCTC18192P.

In addition, the present invention provides a method for producing brazain using the strain.

According to the present invention, after transforming a plasmid expressing brazain into a Lactobacillus reuteri strain, and then treating nisin at a concentration of 30 to 40 ng / ml for 3 to 4 hours, the production of brazain protein is most significant. It is high, and this effect is significantly higher than the brazain produced in the conventional L. lactis strain, the strain of the genus Lactobacillus according to the present invention can be usefully used for producing brazain.

Figure 1 is a figure confirming the Brazin protein transformed in Lactobacillus spp. Strain by PCR using PCR:
Lane 1: 1.1 kb DNA ladder;
Lane 2: 100 bp DNA ladder;
Lane 3: Lactobacillus paracasei KLB8;
Lane 4: Lactobacillus plantarum ) KLB 213; And
Lane 5: Lactobacillus reuteri KCT3594.
Figure 2 shows the results of confirming brazain expression in the genus Lactobacillus over time treatment of nisin:
Figure 2 A is a figure confirming the expression of brazain in Lactobacillus by Western blot;
KLB8: Lactobacillus paracasei ;
KLB 21: Lactobacillus plantarum ;
KCT3594: Lactobacillus reuteri;
2B is a graph in which the image of the western blot is analyzed relatively;
KLB8: Lactobacillus paracasei ;
KLB 21: Lactobacillus plantarum ; And
KCT3594: Lactobacillus reuteri;
Figure 3 is a result confirming the expression of brazain in the genus Lactobacillus according to various concentrations of nisin:
3A is a diagram confirming the expression of brazain at various nisin concentrations by Western blot;
KLB8: Lactobacillus paracasei );
KLB 21: Lactobacillus plantarum );
KCT3594: Lactobacillus reuteri;
3B is a graph in which the image of the western blot is relatively analyzed;
KLB8: Lactobacillus paracasei );
KLB 21: Lactobacillus plantarum ); And
KCT3594: Lactobacillus reuteri.
Figure 4 is another Lactobacillus ( Lactobacillus) spp.) genotype strains and L. lactis in comparison with the expression of brazain:
4A is a Western blot plot showing the expression of brazain in other Lactobacillus strains;
Lane 1: Lactobacillus paracasei ) KLB8;
Lane 2: Lactobacillus plantarum ) KLB 213;
Lane 3: Lactobacillus reuteri KCT3594;
Lane 4: Lactobacillus Nose Rock Athletics Syracuse (. Lactococcus lactis);
4B is a graph in which the image of the western blot is relatively analyzed;
KLB8: Lactobacillus paracasei );
KLB 21: Lactobacillus plantarum );
KCT3594: Lactobacillus reuteri; And
IL1403: Lactobacillus Nose Rock Athletics Syracuse (Lactococcus lactis.).
5 is a graph comparing the stability of pMSP3545 :: Bra-ht in three types of Lactobacillus.

Hereinafter, the present invention will be described in detail.

The present invention provides a Lactobacillus reuteri strain expressing brazain having the amino acid sequence set forth in SEQ ID NO: 1.

The strain is preferably deposited with accession number KCTC18192P, but is not limited thereto. Lactobacillus reuteri expressing brazain according to the present invention was deposited on May 27, 2010 to the Korea Research Institute of Bioscience and Biotechnology (KCTC18192P).

The strain of Lactobacillus expressing the brazain of the present invention is characterized in that pMSP3545 :: Bra-ht plasmid expressing brazain is transferred to Lactobacillus according to the prior art document (Korean. J. Biotechnol. Bioeng 24: 182-188). Preferably, the method is transformed through a perforation method, but is not limited thereto. In addition, the strain of the genus Lactobacillus is preferably Lactobacillus reuteri ( Lactobacillus reuteri ), but is not limited thereto. The transformed Lactobacillus strain is preferably erythromycin resistant, but is not limited thereto. In addition, it is preferable to add 1 to 20 μg / ml of erythromycin, and more preferably to add 3 to 15 μg / ml, It is not limited to this. Nisin added to express brazain in the Lactobacillus strain expressing the brazain is preferably added to the early exponential phase according to the Lactobacillus strain, but is not limited thereto. Nisin added (nisin) is preferably 5 ~ 70 ng / ㎖, it is more preferably added 10 ~ 50 ng / ㎖, but is not limited thereto.

In a specific embodiment of the present invention, after introducing pMSP3545 :: Bra-ht plasmid expressing brazain into the Lactobacillus strain, the expression of brazain was confirmed by colony PCR. As described above, it was confirmed that pMSP3545 :: Bra-ht expressed brazain in the Lactobacillus strain according to the PCR product observed at 500 bp corresponding size of the brazain protein (see FIG. 1).

In addition, in a specific embodiment of the present invention, in order to optimize the expression of brazain protein in the Lactobacillus strain according to the present invention, after comparing the amount of brazain expression according to the treatment time of nisin, after treating the nisin, After 3 to 4 hours, the highest expression level was shown, and the expression level gradually decreased as time passed (see FIG. 2).

In addition, in a specific embodiment of the present invention, in order to optimize the expression level of the Brazain protein in the Lactobacillus sp. Strain according to the present invention, by varying the concentration of the treated nisin, the expression level is compared, 25 ~ 40 ng / The maximum amount was expressed at the concentration of ml, and at higher concentrations, nisin significantly inhibited cell growth, thereby confirming a decrease in the amount of brazain produced (see FIG. 3).

In addition, in a specific embodiment of the present invention, by measuring the expression of brazain using another strain of the genus Lactobacillus, Lb. reuteri It was confirmed that the expression level was the highest in KCTC3594 (see FIG. 4).

In addition, in a specific embodiment of the present invention, as a result of measuring the stability in the strain of the genus Lactobacillus of the plasmid expressing brazain, there is a difference in the expression amount of brazain according to the stability of the strain, the expression amount of brazain high-Lb. The highest stability of the plasmid was in reuteri KCTC3594 (see FIG. 5).

In addition, the present invention provides a method for producing brazain using the Lactobacillus strain according to the present invention.

The method for producing brazain preferably includes, but is not limited to, the following steps:

1) culturing the Lactobacillus reuteri strain deposited with accession number KCTC181921P; And

2) A brazain production method comprising the step of separating the brazain protein from the culture.

In the method for producing brazain, the culture of step 1) is preferably culturing erythromycin in a medium containing 3 to 15 ㎍ / ㎖, in a medium containing 5 to 10 ㎍ / ㎖ More preferably, but not limited to the culture. In addition, the culture of step 1) is preferably cultured in a medium containing 10 to 70 ng / ㎖ nisin (nisin), more preferably in a medium containing 25 to 50 ng / ㎖, but is not limited thereto. It doesn't work. In addition, the culture of step 1) is preferably incubated by treating for 2 to 5 hours, and more preferably for 3 to 4 hours of incubation, but not limited thereto.

In a specific embodiment of the present invention, the strain of Lactobacillus genus that produces brazain has a higher amount of brazain than L. latcis , which produces brazain , and in order to optimize the expression of brazain , By adjusting the treatment time and concentration of the nisin to induce, it was confirmed that the expression level was the highest after 3-4 hours of nisin treatment, and the concentration of nisin added was 25-40 ng / ml. In addition, these expression levels showed a difference depending on the type of strains of the genus Lactobacillus, it was found that the cause is due to the difference in stability in the strains of the genus Lactobacillus of the inserted plasmid (Figs. 1,2,3,4). And 5).

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

< Example  1> Lactobacillus  Transformation of the genus

In order to construct a novel strain expressing sweet protein Brazain, pMSP3545 :: Bra-ht plasmid expressing sweet protein was expressed in "Determination of optimal electroformation conditions for various Lacobacillus spp." (Koran. J. Biotechnol. Bioeng 24:. method in Lactobacillus (Lactobacillus spp according to set forth in 182-188)), Lactobacillus para casei (Lactobacillus paracasei), the electric Lactobacillus Planta room (Lactobacillus plantarum) and Lactobacillus flow Terry (Lactobacillus reuteri) Transformation was performed by electrotransformation.

Lactobacillus strains of the three genera were inoculated in 5 ml of MRS broth (Bioworld) to which 1% glycine (Glycine) was added at 16-18 hours of incubation in the stationary phase. The cells were pelleted by centrifugation at 6,000 g and 4 ° C. for about 5 hours after the inoculation of the initial log phase (OD ₆₀₀ ) of 0.2 to 0.3. The cell pellet was washed twice with cold washing buffer [5 mM sodium phosphate, pH 7.4, 1 mM MgCl ₂ ], followed by electroporation buffer [S buffer, 0.5 M sucrose] )] And the final wash. 1 μl of pMSP3545 :: Bra-ht was mixed with 50 μl of cold cell suspension in a Gene Pulser ^™ disposable cuvette with 0.1 cm internal electrode space and then placed on ice for 5 minutes. The mixture gave electrical stimulation of 12.5 kV / cm peak field strength. After performing electroporation, 200 μl of normal MRS broth was immediately added to the cells, followed by reaction at 37 ° C. for 2 hours. Thereafter, 5 μg / ml of erythromycin was added to the MRS agar plate. The plates were reacted at 37 ° C. for 2-3 hours.

< Experimental Example  1> Sweetness  Expressing protein Lactobacillus  Check

pMSP3545 :: Bra-ht In order to confirm that Lactobacillus spp. Was transformed, amplification of the erythromycin resistance gene of a protein expressing brazain was performed by colony PCR. Confirmed .

To identify the erythromycin resistance gene, a single colony was placed in a 5 × buffer 10 μl, 10 mM dNTP 1 μl, 10 pM forward primer (SEQ ID NO 2: 5′-GAAATTGGAACAGGTAAAGG-3 ′) 1 μl, 10 pM reverse primer (SEQ ID NO: 3: 5'-ATGGGTCAATCGAGAATATC-3 ') 1 μl, Taq polymerase 2 unit and the remaining volume were filled with distilled water and added so that the total volume was 50 μl. PCR conditions were denatured at 94 ° C for 5 minutes, repeated 35 cycles at 94 ° C for 1 minute, 47.7 ° C for 1 minute, and 72 ° C for 1 minute, and then amplified at 72 ° C for the last time.

As a result, as shown in Figure 1, the amplified erythromycin resistance gene showed a size of about 500 bp corresponding to the expected length, through which pMSP3545 :: Bra-ht plasmid expressing sweet protein It was confirmed that all Lactobacillus transformed successfully (Fig. 1).

< Experimental Example  2> Nisin  According to processing time Brazain  Expression change measurement

Transformed in <Example 1> The amount of brazain expression produced in Lactobacillus was measured according to the change in nisin treatment time. It includes pMSP3545 :: Bra-ht transformed in <Example 1> The genus Lactobacillus was diluted in fresh medium at a ratio of 1:50 and incubated for 1 hour 30 minutes to reach an initial exponential phase. Referring to the time required for adaptation of the NICE system described in the previously published report to the culture, 25 ng / ml nisin was added to the initial exponential phase after 1 hour 30 minutes (Pavan et al. 2000). In addition, the culture without adding nisin was used as a negative control. The cultured cells were obtained by centrifugation at 6,000 g for 5 minutes, and the obtained cell pellet was washed twice with 0.1 M phosphate buffer (pH 7.0) and finally with 400 μl of phosphate buffer. Suspended. Cells were then sonicated on ice for 30 minutes with 30% amplification, 30-s pause, 30-s intermediate pause.

In order to compare the expression level of brazain protein in the sonicated cells, western blot was performed. Western blots were performed according to the methods described in Sambrook et al (1989) and Berlec et al (2006). Specifically, the protein sample was mixed with 5 × SDS-PAGE loading buffer and boiled at 100 ° C. for 5 minutes to undergo denaturation. Each protein was loaded onto a 16% SDS-PAGE gel and then transferred to a 0.2 uM PVDF membrane (Bio-Rad). The protein-transported PVDF membrane was blocked for 1 hour at room temperature to prevent nonspecific binding, and reacted with anti-Brazzein antibody overnight at 4 ° C (Berlec et al, 2006). PVDF membrane After washing with TBST buffer [20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween20, pH 7.6], the solution was reacted with HRP-bound goat anti-rabbit IgG (Santacruz) diluted 1: 10,000. . After the reaction, the PVDF membrane was washed and developed on the film (AGFA) using Western blot detection solution (Neronex).

As a result of comparing the expression level of brazain according to the treatment time of nisin, the highest expression level was confirmed 3 to 4 hours after treatment with nisin, and then gradually decreased expression level was observed. As a result of comparing the amount of brazain at the time of not treated with nisin to the negative control group and quantifying Western blot results, as shown in B of FIG. 2, the amount of brazain gradually increased after treatment with nisin 3 to 4 Expressing the highest amount in time, it was confirmed that gradually decreased (Fig. 2). This was presumably due to the hydrolytic capacity of the strain or the decrease in the activity of nisin by adsorption of the resulting cells (Berlec et al, 2006) (FIG. 2).

< Experimental Example  3> Nisin  According to concentration Brazain  Expression difference measurement

To determine the effect of nisin concentration on the expression of brazain in Lactobacillus, after treating various concentrations of nisin (10, 25, 40 and 50 ng / ml), the expression levels of brazain were compared.

The genus Lactobacillus transformed with pMSP3545 :: Bra-ht transformed in <Example 1> was diluted in a fresh medium at a ratio of 1:50, and cultured for 1 hour and 30 minutes until the initial exponential phase. It was. After processing the nisin, the time of the bra Most expression, was determined with reference to the results, whereby the bra expression induction time L. paracasei For KLB 58 4 times, according Lb. plantarum KLB213 and Lb. reuteri KCTC3594 was reacted after treatment with nisin for 3 hours. In addition, a culture without nisin was used as a negative control, and a group treated with 10, 25, 40 and 50 ng / ml of nisin was used as an experimental group. Cells in this culture were obtained by centrifugation at 6,000 g for 5 minutes, and the obtained cell pellet was washed twice with 0.1 M phosphate buffer (pH 7.0) and finally suspended with 400 μl of phosphate buffer. Cells were then sonicated on ice for 30 minutes with 30% amplification, 30-s pause, 30-s intermediate pause.

In order to compare the expression of the brazain protein, Western blots were performed (Sambrook et al (1989) and Berlec et al (2006)). Specifically, the protein sample was mixed with 5 × SDS-PAGE loading buffer, boiled at 100 ° C. for 5 minutes, denatured, and the protein was separated from the 16% SDS-PAGE gel, followed by a 0.2 uM PVDF membrane (Bio-Rad). Moved to. The protein-transported PVDF membrane was blocked for 1 hour at room temperature, and reacted with anti-Brazzein antibody at night at 4 ° C. (Berlec et al, 2006). After washing the PVDF membrane with TBST buffer [20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween20, pH 7.6], diluted HRP-bound goat anti-rabbit IgG (Santacruz) was diluted 1: 10,000. The solution was reacted at room temperature. After the reaction, the PVDF membrane was washed and developed on the film (AGFA) using Western blot detection solution (Neronex).

As a result, when nisin was not added as shown in FIG. 3, the expression of brazain was not observed. When nisin was treated at 25 ng / ml and 40 ng / ml concentrations, the most of brazain was expressed. , Nisin showed a significant inhibitory effect on cell growth when treated at concentrations above 40 ng / ml, which was similar to the results shown in the previously published L. lactis (Berlec et al. 2006) (Fig. 3).

< Experimental Example  4> Lactobacillus  By strain type Brazain  Expression change measurement

The expression levels of brazain were compared in other Lactobacillus spp. Strains and L. lactis IL1403. Lb. paracasei is KLB58 was allowed to react for after treatment of the Nisin 25 ng / ㎖, 4 sigan, Lb. plantarum KLB213 and Lb. reuteri KCTC3594 was treated with 40 ng / ml of nisin and reacted for 3 hours. Cells in each of these cultures were obtained by centrifugation at 6,000 g for 5 minutes, and the obtained cell pellet was washed twice with 0.1 M phosphate buffer (pH 7.0) and finally suspended with 400 μl of phosphate buffer. . Cells were then sonicated on ice for 30 minutes with 30% amplification, 30-s pause, 30-s intermediate pause.

In order to compare the expression of the brazain protein in the samples, Western blots were performed (Sambrook et al (1989) and Berlec et al (2006)). Specifically, the protein sample was mixed with 5 × SDS-PAGE loading buffer, boiled at 100 ° C. for 5 minutes, denatured, and the protein was separated from the 16% SDS-PAGE gel, followed by a 0.2 uM PVDF membrane (Bio-Rad). Moved to. The protein-transported PVDF membrane was blocked for 1 hour at room temperature, and reacted with anti-Brazzein antibody at night at 4 ° C. (Berlec et al, 2006). After washing the PVDF membrane with TBST buffer [20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween20, pH 7.6], the solution was diluted 1: 10,000 with HRP-bound goat anti-rabbit IgG (Santacruz). Reaction at After the reaction was completed, and washed, it was developed on the film (AGFA) using Western blot detection solution (Neronex).

As a result, as shown in Figure 4, Lactobacillus reuteri strain ( Lactobacillus reuteri ) strain KCTC3954 expression level was about 1.6 times higher than other strains, these results are different in the expression level according to the species of the genus Lactobacilli (Lactobacilli) It means you can do it. In addition, the expression levels of brazain in the three Lactobacillus strains in L. lactis that successfully expressed the conventional brazain It was confirmed that it was higher (Berlec et al., 2006) (Fig. 4).

< Experimental Example  5> Plasmid Stability Measurement

Plasmid stability was performed by making some modifications to the method described in "Adaptation of the nisin-controlled expression in Lactobacillus plantarum: a tool to study in vivo biological effects" (Appl. Environ Microbiol. 66: 4427-4442). Three strains of Lactobacillus spp., including pMSP3545 :: Bra-ht, were incubated in MRS broth at 37 ° C. Subcultures were performed by adding 50 μl of the previous culture to 5 ml of new MRS broth, and the ratio of erythromycin resistant colon forming unit (CFU) during 5 subcultures was determined by normal MRS agar plate and 5 μg / ml. Bacterial dilutions were planted in the same number of MRS agar plates containing erythromycin, and measured through the resulting total CFU.

Plasmid stability was measured through the presence of the stability of the inserted plasmid in Lactobacillus. After the fifth subculture, Lb. reuteri KCTC3594 maintained about 60% stability, but little stability was maintained in the other two strains. Therefore, pMSP3545 :: Bra-ht is Lb. was relatively more stable than in reuteri KCTC3594 in two different strains, which was identical with the result of more higher the <Experiment 4> compared to the Lactobacillus flow Terry (Lactobacillus reuteri) two different amount from strain KCTC3594 expression of Bra strains . Thus, it was found that plasmid stability is an important factor affecting the expression level of brazain (FIG. 5).

As seen above, the Lactobacillus reuteri strain producing the brazain of the present invention ( Lactobacillus reuteri ) strain is a strain capable of producing a large amount of sweet protein brazain, by using the sugar substitute brazain It can be useful for mass production.

Korea Research Institute of Bioscience and Biotechnology KCTC18192P 20100527

<110> INHA Industry Partenrship Institute <120> Novel Lactobacillus spp. strains for producing sweet-tasting          protein, Brazzein and a method for producing the Brazzein using          the same <130> 10p-04-04 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> Brazzein <400> 1 atggcgcagg acaaatgtaa aaaagtatac gaaaactacc cggtatccaa atgtcagctg 60 gcaaaccagt gtaactacga ctgtaaactg gacaaacacg ctcgttccgg agaatgcttc 120 tacgacgaaa aacgtaacct gcagtgcatc tgcgactac 159

Claims (6)

A Lactobacillus reuteri strain expressing brazain having an amino acid sequence set forth in SEQ ID NO: 1.
The Lactobacillus reuteri strain according to claim 1, wherein the strain is deposited with accession number KCTC18192P.
1) culturing the strain of claim 1;
2) A brazain production method comprising the step of separating the brazain protein from the culture.
The method of claim 3, wherein the culturing of step 1) is a brazain production method, characterized in that the culture in a medium containing 3 to 15 ㎍ / erythromycin (erythromycin).
The method of claim 3, wherein the culturing of step 1) is a brazain production method, characterized in that the culture in a medium containing 10 to 50 ng / ㎖ of nisin (nisin).
The method of claim 3, wherein the culturing of step 1) is a brazain production method characterized in that the culture by treating the nisin 3 to 4 hours.

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