• Journal of Microbiology and Biotechnology
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• v.27 no.3
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• pp.598-609
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• 2017

Pediococcus pentosaceus ID-7 was isolated from kimchi, a Korean fermented food, and it showed high activity for lactose hydrolysis. The ${\beta}$-galactosidase of P. pentosaceus ID-7 belongs to the GH2 group, which is composed of two distinct proteins. The heterodimeric LacLM type of ${\beta}$-galactosidase found in P. pentosaceus ID-7 consists of two genes partially overlapped, lacL and lacM encoding LacL (72.2 kDa) and LacM (35.4 kDa). In this study, Escherichia coli MM294 was used for the production of LacL, LacM, and LacLM. These three types of recombinant proteins were expressed, purified, and characterized. The specific activities of LacLM and LacL were 339 and 31 U/mg, respectively. However, activity was not detected with LacM alone. The optimal pH of LacLM and LacL was pH 7.5 and pH 7.0, and the optimal temperature of LacLM and LacL was $40^{\circ}C$ and $50^{\circ}C$, respectively. The optimal temperature changes indicate that LacLM is able to achieve higher activity at a relatively lower temperature. LacLM was strongly activated by $Mg^{2+}$, $Mn^{2+}$, and $Zn^{2+}$, which was not true for LacL. Consistent with this, EDTA strongly inactivated LacLM and LacL, but the presence of reducing agents did not dramatically alter the activity. Taken together, multiple alignment of amino acid sequences and phylogenetic analysis results of LacL and LacM of P. pentosaceus ID-7 suggest the evolution of LacL into LacLM and that the use of divalent metal ions results in higher activity.

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.550-558
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• 2021

Lactobacillus kefiranofaciens contains two types of β-galactosidase, LacLM and LacZ, belonging to different glycoside hydrolase families. The difference in function between them has been unclear so far for practical application. In this study, LacLM and LacZ from L. kefiranofaciens ATCC51647 were cloned into constitutive lactobacillal expression vector pMG36e, respectively. Furtherly, pMG36n-lacs was constructed from pMG36e-lacs by replacing erythromycin with nisin as selective marker for food-grade expressing systems in Lactobacillus plantarum WCFS1, designated recombinant LacLM and LacZ respectively. The results from hydrolysis of o-nitrophenyl-β-galactopyranoside (ONPG) showed that the β-galactosidases activity of the recombinant LacLM and LacZ was 1460% and 670% higher than that of the original L. kefiranofaciens. Moreover, the lactose hydrolytic activity of recombinant LacLM was higher than that of LacZ in milk. Nevertheless, compare to LacZ, in 25% lactose solution the galacto-oligosaccharides (GOS) production of recombinant LacLM was lower. Therefore, two β-galactopyranosides could play different roles in carbohydrate metabolism of L. kefiranofaciens. In addition, the maximal growth rate of two recombinant strains were evaluated with different temperature level and nisin concentration in fermentation assay for practical purpose. The results displayed that 37℃ and 20-40 U/ml nisin were the optimal fermentation conditions for the growth of recombinant β-galactosidase strains. Altogether the food-grade Expression system of recombinant β-galactosidase was feasible for applications in the food and dairy industry.

• Journal of Microbiology and Biotechnology
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• v.26 no.1
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• pp.20-27
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• 2016

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60℃ and 7.0, and 50℃ and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

• Microbiology and Biotechnology Letters
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• v.48 no.3
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• pp.287-295
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• 2020

Lactobacillus zymae GU240 was previously isolated from Kimchi, a Korean fermented vegetable, as a strong GABA producer. The strain showed β-galactosidase (β-Gal) activity on MRS agar plates with X-gal. When growth and β-Gal activities of GU240 were measured using MRS (glucose, 2%, w/v) and MRSL (lactose, 2%, w/v) broths, cells were found to grow slowly in MRSL, and the β-Gal activity (36 units at 4 h) was lower than that of cells grown in MRS (94 units at 16 h). The highest OD₆₀₀ value of the culture in MRS was 1.6 at 24 h at 37℃, whereas that of the culture in MRSL was 0.6 at 16 h. β-Gal activity of the culture in MRS reached the maximum (95.6 u/ml) at 16 h, decreased thereafter, and was not detected at 48 h. β-Gal activity for culture in MRSL reached its highest (36 u/ml) at 4 h and decreased gradually, but some activity (11.05 u/ml) still remained at 72 h. The structural gene encoding β-Gal in L. zymae GU240 was cloned as a 3.1 kb fragment, and DNA sequencing confirmed the presence of complete lacLM genes. lacLM genes from L. zymae GU240 showed 98-99% homologies in nucleotide sequences with other lacLM genes from L. brevis. Reverse transcription (RT)-PCR confirmed the operon structure of lacLM. The results indicated that L. zymae GU240 might be in the process of losing the ability to grow rapidly on lactose-containing medium, such as milk, due to adaptations to plant environments, including kimchi.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.241-247
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• 2005

A new lactococcal shuttle/expression vector for lactococci, pWgal13T, was constructed using a $\beta$-galactosi-dase gene (lacZ) from Lacfococcus lactis ssp. lactis ATCC 7962 as a screening marker. The pWgal 13T was introduced into Escherichia coli DH5a and L. lactis MG1363, and was easily detected by the formation of blue colonies on a medium containing X-gal without any false transformants. Also, the quantitatively lacZ activity of pWgal13T was measured in L. lactis ssp. cremoris MG1363, and was found to be four times higher than that of L. lactis ssp. lactis ATCC7962 grown on a medium containing glucose, which shows that the lacZ gene of pWgal13T can be used for the efficient screening of L. lactis on general media. The pWgal13T was equipped with a lactococcal replicon of pWV01 from L. lactis Wg2, the new promoter P13C from L. lactis ssp. cremoris LM0230, multiple cloning sites, and a terminator for the expression of a relevant gene. The vee-tor pWgal13T was used for the expression of the EGFP gene in E. coli and L. lactis. These results show that the lactococcal expression/shuttle vector constructed in the present study can be used for the production of foreign proteins in E. coli and L. lactis.

• Microbiology and Biotechnology Letters
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• v.33 no.1
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• pp.41-50
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• 2005

An organic acid tolerance mutant (M-200) was obtained from Leuconostoc mesenteroides KCCM 35471, followed by the screening procedure using a specific organic acid medium (lactic acid: acetic acid, 2:1). The characteristics of the acid tolerance M-200 and the wild type LM-W were examined at various temperature and pH ranges $(l0-30^{\circ}C$ of temp, 3.5-4.5 of pH). The growth of strain M-200 at HCl adjusted medium $(10^{\circ}C\;and\;pH 3.5)$ was observed. In the case of organic acid adjusted medium, the strain showed its growth at the pH range of 3.8. When the strain M-200 was used as a starter for Kimchi fermentation, a constant acid level (0.55) was observed during the whole fermentation period. This result indicates that the strain produces a proper level of acid content for the Kimchi fermentation. This result also indicates that the edible period of Kimchi can be extended to 3.5 fold compare to the result obtained from the LM-W used Kimchi fermentation. However the excess use of the strain M-200 showed the inhibition of growth of Lactobacillus plantarum, low lactic acid level content and low level of organoleptic test. In the case of organic acid content during the Kimchi fermentation, the strain M-200 showed relatively low production rate compare to the wild type (M-200: 3.5 mg/L at 21 days of fermentation, LM-W: 7 mg/L at 21 days of fermentation). Therefore a mixed Kimchi starter containing M-200 and other strains probably maintain a good Kimchi quality during the fermentation.

• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.389-398
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• 2009

Development of expression vectors is important for the basic and applied researches on kimchi LAB (lactic acid bacteria). An expression vector, pSJE6c was constructed by inserting P6C promoter sequence from Lactococcus lactis into pSJE, a shuttle vector for E. coli and Leuconostoc species. To test the efficiency of pSJE6c, aga ($\alpha$-galactosidase) and lacZ ($\beta$-galactosidase) genes were expressed in Lactobacillus brevis 2.14. Compared to the pSJE, expression levels of both genes were increased, indicating P6C promoter was better than indigenous promoters. Enzyme activities of L. brevis cells harboring pSJE6caga (pSJE6c with aga) or pSJE6Z (pSJE6c with lacZ) were 1.5-2 fold higher than those with pSJEaga (pSJE with aga) or pSJEZ (pSJE with lacZ). More RNA transcripts were detected in cells harboring pSJE6c based recombinant plasmid. The results indicated that heterologous gene expressions in kimchi LAB could be improved significantly by use of efficient expression vectors.

• Journal of Animal Science and Technology
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• v.47 no.4
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• pp.655-666
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• 2005

This study was performed to measure physicochemical and textural characteristics, and shelf-life effect of low-fat functional sausages(LFFSs) manufactured with sodium lactate(SL, 3.3%), lac pigment and various molecular weights(MWs) of chitosan (Low=1.5 kDa, Med=30-50 kDa and High=200 kDa) during storage at 15$^{\circ}C$ for 18 days. LFFSs had 73.7-76.0% moisture, lower than 3% fat and 14-15% protein, respectively. pH values were 6.05-6.44 and the control(150 ppm, $NaNO_2$) was the lowest among LFFSs (p<0.05). Increasing storage time decreased pH values, but no differences in pH values were observed up to 6 days of storage (p>0.05). LFFSs containing SL and low MW of chitosan improved water holding capacity (WHC) and different from those with SL and medium-MW chitosan. WHC was decreased with increased storage time and differences of WHC were observed from 18 days of storage. The addition of chitosan reduced both lightness and redness values, as compared to 150 ppm sodium nitrite(SN), and increased storage time decreased yellowness(p<0.05), especially at 12 days of storage. LFFSs with SL and medium-MW chitosan increased most textural properties compared to the control(p<0.05). The addition of SN of 150 ppm in LFFSs retarded microbial growth for E. coli 0157:H7, while those with SL tended to have an antimicrobial effect for Listeria monocytogenes(LM). The growth rate of LM was delayed by addition of various MW of chitosans in LFFSs, especially high MW chitosan, as compared to LFFSs containing SL alone. These results indicated that the functional, textural and antimicrobial effects of LFFSs were improved by addition SL and various MW of chitosan combinations. In addition, 0.05% lac pigment improved the cure color of LFFSs similar to those of 150 ppm SN.

• Food Science and Preservation
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• v.22 no.5
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• pp.758-767
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• 2015

Red pepper powder sensitive to aflatoxins contamination is major ingredients of kimchi. This study was conducted to investigate the effect of lactic acid bacteria (LAB) on the changes in aflatoxin levels during kimchi fermentation. Baechu kimchi was contaminated with aflatoxins ($B_1$, $B_2$, $G_1$, and $G_2$) and inoculated with LAB (Lactobacillus plantarum and Leuconostoc mesenteroides), and the following characteristics were investigated for 8 weeks: pH, titratable acidity, salinity, microbial properties, and aflatoxin levels. The pH decreased rapidly during storage, and the titratable acidity was increased. The salinity of the samples was shown to increase from 2.30 to 2.40%. The total number of aerobic bacteria and lactic acid bacteria in kimchi inoculated with LAB was significantly higher than that of the others. Yeast and molds were detected at approximately 1~3 log CFU/g during storage. Coliforms were detected in the control after 4 weeks, whereas in other samples they were not detected until after 2 weeks. The aflatoxin levels reduced during kimchi fermentation. The average reduction rate of aflatoxin levels during kimchi fermentation was 8.39%, but in kimchi inoculated with Lac. plantarum and Leu. mesenteroides, the rate were 25.16 % and 27.86%, respectively. These results showed that aflatoxins can be removed by LAB during kimchi fermentation.

• Food Science of Animal Resources
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• v.23 no.2
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• pp.128-136
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• 2003

This study was peformed to evaluate physico-chemical and textural properties, and shelf-life effect of low-fat functional sausages(LFFS) manufactured with sodium lactate(SL), lac color and various molecular weights of chitosans(low=1.5 kDa, medium=30∼40 kDa and high=200 kDa) during storage at 4$^{\circ}C$ for 8 weeks. LFFS had a pH range of 6.39∼6.50, 76∼78% moisture, <2% fat, 14∼15% protein. The combination of SL and low molecular weight(MW) of chitosan improved water holding capacity(WHC), however those of SL and medium MW of chitosan reduced WHC. Vacuum purge(VP) reduced with increased MW of chitosans during refrigerated storage. The addition of chitosans reduced the lightness and yellowness, but increased the redness values, which was comparable to the sodium nitrite concentration between 75 and 150 ppm. LFFS containing SL and medium MW of chitosan increased most texture profile analysis(TPA) values, as compared to controls with 75 and 150 ppm. The addition of SL in LFFS retarded the microbial growth for Listeria monocytogenes, however no synergistic effect with the addition of chitosans were observed. E coli O157:H7 and Salmonella typhimurium reduced during refrigerated storage, regardless of SL and chitosan treatments. Increased storage time increased values for VP, yellowness and textural properties. These results indicated that the combination of SL and various MW of chitosans affected the functional and textural properties, and inhibited the microbial growth for LM effectively. In addition, 0.5% lac color as a replacer for sodium nitrite improved the color development, resulting in similar hunter color values, which was comparable to the sodium nitrite concentration between 75 and 150 ppm.