• Journal of Animal Science and Technology
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• 제63권1호
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• pp.191-193
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• 2021

Lactococcus lactis is a fermentative lactic acid bacterium that is used extensively in food fermentations. The L. lactis strain K_LL005 was isolated from the grasshopper (Oxya chinensis sinuosa) gut in Korea. In this study, we reported the complete genome sequence of Lactococcus lactis K_LL005. The final complete genome assembly consist of one circular chromosome (2,375,093 bp) with an overall guanine + cytosine (G + C) content of 35.0%. Annotation results revealed 2,281 protein-coding sequences (CDSs), 19 rRNAs, and 68 tRNA genes. Lactococcus lactis K_LL005 has a gene encoding xylose metabolism such as xylR, xylA, and xylB (xylRAB).

• 한국축산식품학회지
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• 제42권1호
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• pp.46-60
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• 2022

In this study, angiotensin-I-converting enzyme inhibitory (ACEI) activity was evaluated in fermented goat milk fermented by lactic acid bacteria (LAB) from fermented foods and breast milk. Furthermore, the potential for ACEI peptides was identified in fermented goat milk with the highest ACEI activity. The proteolytic specificity of LAB was also evaluated. The 2% isolate was inoculated into reconstituted goat milk (11%, w/v), then incubated at 37℃ until pH 4.6 was reached. The supernatant produced by centrifugation was analyzed for ACEI activity and total peptide. Viable cell counts of LAB and titratable acidity were also evaluated after fermentation. Peptide identification was carried out using nano liquid chromatography mass spectrometry (LC-MS/MS), and potential as an ACEI peptide was carried out based on a literature review. The result revealed that ACEI activity was produced in all samples (20.44%-60.33%). Fermented goat milk of Lc. lactis ssp. lactis BD17 produced the highest ACEI activity (60.33%; IC₅₀ 0.297±0.10 mg/mL) after 48 h incubation, viable cell counts >8 Log CFU/mL, and peptide content of 4.037±0.27/mL. A total of 261 peptides were released, predominantly derived from casein (93%). The proteolytic specificity of Lc. lactis ssp. lactis BD17 through cleavage on the amino acid tyrosine, leucine, glutamic acid, and proline. A total of 21 peptides were identified as ACEI peptides. This study showed that one of the isolates from fermented food, namely Lc. lactis ssp. lactis BD17, has the potential as a starter culture for the production of fermented goat milk which has functional properties as a source of antihypertensive peptides.

• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.154-162
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• 2021

L-5-methyltetrahydrofolate (5-MTHF) is one of the biological active forms of folate, which is widely used as a nutraceutical. However, low yield and serious pollution associated with the chemical synthesis of 5-MTHF hampers its sustainable supply. In this study, 5-MTHF production was improved by engineering the 5-MTHF biosynthesis pathway and NADPH supply in Lactococcus lactis for developing a green and sustainable biosynthesis approach. Specifically, overexpressing the key rate-limiting enzyme methylenetetrahydrofolate reductase led to intracellular 5-MTHF accumulation, reaching 18 ㎍/l. Next, 5-MTHF synthesis was further enhanced by combinatorial overexpression of 5-MTHF synthesis pathway enzymes with methylenetetrahydrofolate reductase, resulting in 1.7-fold enhancement. The folate supply pathway was strengthened by expressing folE encoding GTP cyclohydrolase I, which increased 5-MTHF production 2.4-fold to 72 ㎍/l. Furthermore, glucose-6-phosphate dehydrogenase was overexpressed to improve the redox cofactor NADPH supply for 5-MTHF biosynthesis, which led to a 60% increase in intracellular NADPH and a 35% increase in 5-MTHF production (97 ㎍/l). To reduce formation of the by-product 5-formyltetrahydrofolate, overexpression of 5-formyltetrahydrofolate cyclo-ligase converted 5-formyltetrahydrofolate to 5,10-methyltetrahydrofolate, which enhanced the 5-MTHF titer to 132 ㎍/l. Finally, combinatorial addition of folate precursors to the fermentation medium boosted 5-MTHF production, reaching 300 ㎍/l. To the best of our knowledge, this titer is the highest achieved by L. lactis. This study lays the foundation for further engineering of L. lactis for efficient 5-MTHF biosynthesis.

• 한국미생물·생명공학회지
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• 제49권1호
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• pp.75-87
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• 2021

Type I Interferons (IFNα) are known for their role as biological anticancer agents owing to their cell-apoptosis inducing properties. Development of an appropriate, cost-effective host expression system is crucial for meeting the increasing demand for proteins. Therefore, this study aims to develop codon-optimized IFNα-2b in L. lactis NZ3900. These cells express extracellular protein using the NICE system and Usp₄₅ signal peptide. To validate the mature form of the expressed protein, the recombinant IFNα-2b was screened in a human colorectal cancer cell line using the cytotoxicity assay. The IFNα-2b was successfully cloned into the pNZ8148 vector, thereby generating recombinant L. lactis pNZ8148-SP_Usp45-IFNα-2b. The computational analysis of codon-optimized IFNα-2b revealed no mutation and amino acid changes; additionally, the codon-optimized IFNα-2b showed 100% similarity with native human IFNα-2b, in the BLAST analysis. The partial size exclusion chromatography (SEC) of extracellular protein yielded a 19 kDa protein, which was further confirmed by its positive binding to anti-IFNα-2b in the western blot analysis. The crude protein and SEC-purified partial fraction showed IC₅₀ values of 33.22 ㎍/ml and 127.2 ㎍/ml, respectively, which indicated better activity than the metabolites of L. lactis NZ3900 (231.8 ㎍/ml). These values were also comparable with those of the regular anticancer drug tamoxifen (105.5 ㎍/ml). These results demonstrated L. lactis as a promising host system that functions by utilizing the pNZ8148 NICE system. Meanwhile, codon-optimized usage of the inserted gene increased the optimal protein expression levels, which could be beneficial for its large-scale production. Taken together, the recombinant L. lactis IFNα-2b is a potential alternative treatment for colorectal cancer. Furthermore, its activity was analyzed in the WiDr cell line, to assess its colorectal anticancer activities in vivo.

• 한국축산식품학회지
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• 제40권2호
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• pp.155-171
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• 2020

Fermented products, including sausages, provide several health benefits, particularly when probiotics are used in the fermentation process. This study aimed to examine the cytotoxicity (against Caco-2 and MCF-7 cell lines), antihypertensive activity via angiotensin-converting enzyme (ACE) inhibition, antioxidant capacity, antidiabetic activity via α-amylase and α-glucosidase inhibition, proteolysis rate, and oxidative degradation of fermented camel and beef sausages in vitro by the novel probiotic Lactococcus lactis KX881782 isolated from camel milk. Moreover, camel and beef sausages fermented with commercial starter culture alone were compared to those fermented with commercial starter culture combined with L. lactis. The degree of hydrolysis, antioxidant capacity, cytotoxicity against Caco-2 and MCF-7, α-amylase, α-glucosidase, and ACE inhibitory activities were higher (p<0.05) in fermented camel sausages than beef sausages. In contrast, the water and lipid peroxidation activity were lower (p<0.05) in camel sausages than beef sausages. L. lactis enhanced the health benefits of the fermented camel sausages. These results suggest that camel sausage fermented with the novel probiotic L. lactis KX881782 could be a promising functional food that relatively provides several health benefits to consumers compared with fermented beef sausage.

• Applied Biological Chemistry
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• 제46권4호
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• pp.367-375
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• 2003

복분자 과육의 농축액을 유산균을 이용하여 발효시킨 후 발효액의 생리활성을 평가하였다. 발효에는 Lactobacillus acidophilus KCCM 32820, L. casei KCCM 12452, Lactococcus lactis subsp. lactis KCCM 40104, Streptococcus thermophilus KCCM 40430을 단독 또는 혼합하여 사용하였으며 접종량은 대수증식기 말기의 배양액을 2%(v/v)가 되도록 첨가하였다. 단독발효의 경우 L. casei의 발효능이 가장 우수하였으며 혼합 starter를 사용하였을 경우에는 L. casei와 L. lactis를 1:1로 혼합하였을 때 가장 우수한 발효능을 나타내었으나 관능검사에 있어서 L. acidophilus와 S. thermophilus를 이용하였을 때 종합적 기호도가 가장 높았다. 발효는 올리고당을 1%(w/v) 첨가하고 pH를 4.0, 발효온도를 $35{\sim}37^{\circ}C$로 하였을 때 $72{\sim}96$시간에서 가장 잘 이루어졌다. 발효액에는 glucose와 fructose가 주요 유리당으로 존재하였고 lactic acid 함량은 698.2 mg/100 g으로 발효전보다 9배 이상 증가하였다. 발효액의 생리활성을 측정한 결과 69%의 전자공여효과를 나타내었으며 아질산염 소거기능은 pH 1.2에서 38.3%, SOD 유사활성과 xanthine oxidase 저해활성은 각각 60.3%와 41.8%의 활성을 나타내었다. 발효액은 Escherichia coli 0-157:H7에 대해서는 17.3%의 생육저해율을 나타내 사용한 검정균 중에서 가장 높은 항균력을 보였으며 Salmonella typhimurium과 Bacillus cereus에 대해서는 각각 8.9%, 9.7%의 생육저해효과를 나타내었고 Staphylococcus aureus에 대해서는 7.2%의 생육저해효과를 나타내었다.

• 한국미생물·생명공학회지
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• 제46권2호
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• pp.91-101
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• 2018

The aim of this study was to transfer the 18.5 kb gene clusters coding for 17 genes from Lactobacillus rhamnosus to Lactococcus lactis subsp. cremoris MG1363 in order to determine the effect of host on exopolysaccharide (EPS) production and to provide a model for studying the phosphorylation of proteins which are proposed to be involved in EPS polymerization. Lactobacillus rhamnosus RW-9595M and ATCC 9595 have 99% identical operons coding for EPS biosynthesis, produced different amounts of EPS (543 vs 108 mg/l). L. lactis subsp. cremoris MG1363 transformed with the operons from RW-9595M and ATCC 9595 respectively, produced 326 and 302 mg/l EPS in M17 containing 0.5% glucose. The tyrosine protein kinase transmembrane modulator (Wzd) was proposed to participate in regulating chain elongation of EPS polymers by interacting with the tyrosine protein kinase Wze. While Wzd was found in phosphorylated form in the presence of the phosphorylated kinase (Wze), no phosphorylated proteins were detected when all nine tyrosines of Wzd were mutated to phenylalanine. Lactococcus lactis subsp. cremoris could produce higher amounts of EPS than other EPS-producing lactococci when expressing genes from L. rhamnosus. Phosphorylated Wzd was essential for the phosphorylation of Wze when expressed in vivo.

• 한국정보기술응용학회:학술대회논문집
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• 한국정보기술응용학회 2006년도 춘계학술대회
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• pp.185-207
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• 2006

산업단지는 지난 30년간 한국 산업의 성장을 이끌어 온 발전모형으로서 존재하여 왔으나, 최근 지식에 기초한 혁신창출형 경제체제가 국가 및 지역사회의 경쟁력을 위한 핵심요소로 부각되면서 '효율성' 측면에서 그 의미가 크게 퇴색되었다. 이를 위한 대안으로서 '클러스터'가 대두되어 다양한 분석연구가 수행되고 있으며, 정부와 지방자치단체들은 이를 바탕으로 각자의 특색에 맞는 클러스터 조성 정책을 펼치고 있다. 그러나, 기존의 연구들은 클러스터의 종류 및 발전단계에 관한 프레임워크 제시 등의 이론적 수준에 국한되어 있거나, 지역사례 연구를 통한 성공요인분석(CFS) 및 단순한 정책방향 제시 수준에 머물러 있는 한계를 보이고 있다. 본 연구는 '클러스터'에 관한 선행연구를 분석해 보고, 클러스터의 중요한 판단기준이 되는 군집도와 네트워크 연계 정도를 기준으로 한 '$2{\times}2$ 클러스터 전략격자모형'을 효과적인 클러스터 구축전략 수립을 위한 이론적 틀로서 제시하였다. 또한, 분석틀에 실질적인 사례로서 '충북지역의 SW산업'을 전략격자모형에 대응시켜 분석함으로써 전략격자의 유용성을 제시하였다. 이를 위해, 충북지역의 SW 공급업체와 수요업체를 대상으로 설문조사를 실시, 분석한 후 그 결과를 전략격자모형에 대응시켰다. 그 결과, 충북지역의 SW산업은 아직 산업단지 수준에 있는 것으로 분석되었고 충북의 SW산업의 충북 내의 수요만으로는 더 큰 성장이 어려운 것으로 분석, 지역 내에서의 수요창출을 목표로 하는 '단일 클러스터' 구축보다는 지역적 제약을 벗어난 '매가 클러스터'의 구축으로 지역 내외에서의 수요창출이 가능한 클러스터의 구축을 그 대안으로 제시하였다.${\alpha}$에 E. coli Jm109의 plasmid pBX19, pBR322를 전이시켰다. 6. L. lactis ssp. lactis 균주에 lysozyme 처리시 30${\sim}$80%의 생존율을 보였으며, 대부분의 L. acidophilus 균주의 경우 약 70%의 생존율을 보였다. L. casei 102S의 경우는 45분간 처리 시에도 100%의 생존율을 보였다. 8. L. lactis ssp. lactis 균주에 pLZ12를 6.0kV에서 전이시킨 결과 12.5kV에서보다 형질전환 효율이 훨씬 높았으며 lysozyme 처리에 의해 형질전환 효율이 증가되었다. 9. L. acidophilus 균주에 pLZ12를 전이시 6.0kV에서는 전이가 모두 이루어졌으나, 12.5kV에서는 L. acidophilus WIESBY와 NCFM에서 전이가 이루어지지 않았으며, lysozyme 처리 후 pLZ12를 전이시켰을 때 12kV보다 6.0kV에서 형질전환 효율이 증가되었다. 10. Gene Pulser와 Progenitor II를 사용하여 pLZ12를 L. lactis ssp. lactis 균주에 전이하였을 때 Gene Pulser에 비해 Progenitor II의 형질전환 효율이 현저히 떨어졌다. L. acidophilus HY7008과 HY7001은 두 기기 모두 형질전환이 이루어졌으나, L. acidophilus WEISBY와 NCFM은 Progeni-tor II에서 전이가 일어나지 않았으며, Gene Pulser에서 전이균주를 얻어 두 electroporator간에 형질전환 효율의 차이를 보였다. 11. L. casei 102S에 pLZ12

• Journal of Applied Biological Chemistry
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• 제62권3호
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• pp.257-264
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• 2019

Rice bran is considered a natural source of antioxidants. In this study, rice bran was fermented with lactic acid bacteria to increase its antioxidant activity. Four strains isolated from fermented food, Lactobacillus plantarum MJM60383, Lactococcus lactis subsp. lactis MJM60392, Lactobacillus fermentum MJM60393, and Lactobacillus paracasei MJM60396, were confirmed as safe through stability tests such as safety assessment for biogenic amine production, hemolytic activity, and mucin degradation, and showed high reducing capacity. The antioxidant activity of rice bran fermentation altered by these strains was evaluated using several methods including measurement of $Fe^{2+}$ chelating activity and scavenging activity by 1,1-diphenyl-2-picryl-hydrazil (DPPH), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and nitric oxide assays. In this study, the total phenolic content and ${\gamma}$-oryzanol were evaluated by high-performance liquid chromatography. Compared to non-fermented rice bran and a commercial product, rice bran fermented with Lactococcus lactis subsp. lactis MJM60392 showed the highest phenolic content (844.13 mg GAE/g). Moreover, the content of ferulic acids, ${\rho}$-coumaric acid, and ${\gamma}$-oryzanol in rice bran increased after fermentation with L. lactis subsp. lactis MJM60392 and L. fermentum MJM60393 compared to other samples. Indeed, the DPPH radical scavenging activity and NO scavenging activity were also found to be high in these fermented rice brans. These results indicated that fermentation with lactic acid bacteria increases the active compound levels and the potent antioxidant activities of rice bran.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.829-835
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• 2000

The ptsH and ptsI genes of Lactococus lactis subsp. lactis ATCC 7962 (L. lactis 7962), encoding the general proteins of phosphotransferase system (PTS) components, HPr and enzyme I, respectively, were cloned and characterized. A 1.3 kb PCR product was obtained using a primer set that was hybridized to the internal region of the L. lactis 7962 pts HI genes and then subcloned into a low-copy number vector, pACYC184. The 5' upstream and 3' downstream region from the 1.3 kb fragment were subsequently clone using the chromosome walking method. The complete ptsHI operon was constructed and the nucleotide sequences determined. Two ORFs corresponding to HPr (88 amino acids) and enzyme I (575 amino acids) were located. The ptsHI genes of L. lactis 7962 showed a very high homology (84-90%) with those genes from other Gram-positive bacteria. A primer extension analysis showed that the transcription started at either one of two adjacent bases upstream of the start codon. Using a Northern analysis, two transcripts were detected; the first, a 0.3 kb transcript corresponding to ptsH and the second, a 2 kb transcript corresponding to ptsH and ptsI. The transcription level of ptsH was higher than that of ptsI. The concentration of the ptsH transcript in cells grown on glucose was similar to that in cells grown on lactose, yet higher than that in cells grown on galactose. The ptsI transcript was scarcely detected in cell grown on lactose or galactose. The ptsI transcript was scarcely detected in cells grown on lactose or galactose. The results of a sequence analysis and Northern blot confirmed that the ptsH and ptsI genes of L. lactis 7962 were arranged in an operon like other known ptsHI genes and the expression of the ptsHI genes was regulated at the transcriptional level in response to the carbon source.