• Microbiology and Biotechnology Letters
• /
• v.49 no.1
• /
• pp.75-87
• /
• 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.

• Food Science of Animal Resources
• /
• v.29 no.5
• /
• pp.640-646
• /
• 2009

Lactic acid bacteria (LAB) are typical probiotic microbes that are used in various industries including fermented foods, feed additives, and pharmaceuticals. The purpose of this study was to compare the ability of isoflavone biotransformation and antioxidative activity of 17 LAB. Six strains including the Lactobacillus species exhibited a 100% hydrolysis rate for daidzein during fermentation. The content of total genistein in soymilk fermented with these strains was $872-943\;{\mu}g/g$. The DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging ability of the LAB was widely variable and ranged from 23-78%. A selected strain was isolated from kimchi and the strain was identified as Lactobacillus plantarum ssp. through the API carbohydrate fermentation pattern and 16S rDNA profile. The strain exhibited excellent acid tolerance in an artificial gastric solution. L. plantarum YS712 showed high $\beta$-glucosidase activity in fermentation. The concentration of genistein and daidzein in soymilk fermented with L. plantarum YS712 increased from 3.64 to $917.3\;{\mu}g/g$ and from 58.18 to $1062.17\;{\mu}g/g$, respectively. These results demonstrate the potential of L. plantarum YS712 as a probiotic culture that can be utilized in the manufacturing of fermentation foods and dietary supplements.

• Asian-Australasian Journal of Animal Sciences
• /
• v.28 no.4
• /
• pp.511-518
• /
• 2015

Calliandra calothyrsus preserved in silage is an alternative method for improving the crude protein content of feeds for sustainable ruminant production. The aim of this research was to evaluate the quality of silage which contained different levels of C. calothyrsus by examining the fermentation characteristics and microbial diversity. Silage was made in a completely randomized design consisting of five treatments with three replications i.e.: R0, Pennisetum purpureum 100%; R1, P. purpureum 75%+C. calothyrsus 25%;, R2, P. purpureum 50%+C. calothyrsus 50%; R3, P. purpureum 25%+C. calothyrsus 75%; and R4, C. calothyrsus 100%. All silages were prepared using plastic jar silos (600 g) and incubated at room temperature for 30 days. Silages were analyzed for fermentation characteristics and microbial diversity. Increased levels of C. calothyrsus in silage had a significant effect (p<0.01) on the fermentation characteristics. The microbial diversity index decreased and activity was inhibited with increasing levels of C. calothyrsus. The microbial community indicated that there was a population of Lactobacillus plantarum, L. casei, L. brevis, Lactococcus lactis, Chryseobacterium sp., and uncultured bacteria. The result confirmed that silage with a combination of grass and C. calothyrsus had good fermentation characteristics and microbial communities were dominated by L. plantarum.

• Asian-Australasian Journal of Animal Sciences
• /
• v.26 no.9
• /
• pp.1304-1312
• /
• 2013

The effects of storage period and aerobic deterioration on the bacterial community were examined in Italian ryegrass (IR), guinea grass (GG), and whole-crop maize (WM) silages. Direct-cut forages were stored in a laboratory silo for 3, 7, 14, 28, 56, and 120 d without any additives; live counts, content of fermentation products, and characteristics of the bacterial community were determined. 2,3-Butanediol, acetic acid, and lactic acid were the dominant fermentation products in the IR, GG, and WM silages, respectively. The acetic acid content increased as a result of prolonged ensiling, regardless of the type of silage crop, and the changes were distinctively visible from the beginning of GG ensiling. Pantoea agglomerans, Rahnella aquatilis, and Enterobacter sp. were the major bacteria in the IR silage, indicating that alcoholic fermentation may be due to the activity of enterobacteria. Staphylococcus sciuri and Bacillus pumilus were detected when IR silage was spoiled, whereas between aerobically stable and unstable silages, no differences were seen in the bacterial community at silo opening. Lactococcus lactis was a representative bacterium, although acetic acid was the major fermentation product in the GG silage. Lactobacillus plantarum, Lactobacillus brevis, and Morganella morganii were suggested to be associated with the increase in acetic acid due to prolonged storage. Enterobacter cloacae appeared when the GG silage was spoiled. In the WM silage, no distinctive changes due to prolonged ensiling were seen in the bacterial community. Throughout the ensiling, Weissella paramesenteroides, Weissella confusa, and Klebsiella pneumoniae were present in addition to L. plantarum, L. brevis, and L. lactis. Upon deterioration, Acetobacter pasteurianus, Klebsiella variicola, Enterobacter hormaechei, and Bacillus gibsonii were detected. These results demonstrate the diverse bacterial community that evolves during ensiling and aerobic spoilage of IR, GG, and WM silages.

• Journal of Korean Medicine for Obesity Research
• /
• v.15 no.2
• /
• pp.123-130
• /
• 2015

Objectives: Samjung-hwan (SJH), a well-known traditional fermented herb formula recorded in Dongui Bogam, has been commonly used for prolonging life for four hundred years in Eastern Asia. However, fermented SJH has not been investigated in terms of microbial ecology until present time. Methods: SJH was fermented for five weeks and fermentation characteristics during SJH fermentation were performed including pH, acidity and microbial profiling. Also, we measured total polyphenol and total flavonoid contents and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. In order to select starter candidate, several lactic acid bacteria were isolated from fermented SJH. Results: pH of fermented SJH was decreased from 4.7 to 3.0 and acidity was increased from 0.45% to 1.72%. Also, fermented SJH increased antioxidant indicator such as total polyphenol and total flavonoid as well as DPPH free radical scavenging activity. Lactobacillus brevis was increased, Pseudanabaena sp. was decreased, and Lactococcus lactis subsp. lactis was stable during 5-week fermentation of SJH. L. brevis and L. plantarum were isolated from fermented SJH. Conclusions: Fermented SJH for four weeks had optimal effect on antioxidant and fermentation characteristics such as pH, acidity and microbial profile. Further studies are required to develop starter and analyze functional compounds in oder to produce standardized SJH.

• Journal of Microbiology
• /
• v.43 no.4
• /
• pp.345-353
• /
• 2005

The complex ecosystem of intestinal micro flora is estimated to harbor approximately 400 different microbial species, mostly bacteria. However, studies on bacterial colonization have mostly been based on culturing methods, which only detect a small fraction of the whole microbiotic ecosystem of the gut. To clarify the initial acquisition and subsequent colonization of bacteria in an infant within the few days after birth, phylogenetic analysis was performed using 16S rDNA sequences from the DNA iso-lated from feces on the 1st, 3rd, and 6th day. 16S rDNA libraries were constructed with the amplicons of PCR conditions at 30 cycles and $50^{\circ}C$ annealing temperature. Nine independent libraries were produced by the application of three sets of primers (set A, set B, and set C) combined with three fecal samples for day 1, day 3, and day 6 of life. Approximately 220 clones ($76.7\%$) of all 325 isolated clones were characterized as known species, while other 105 clones ($32.3\%$) were characterized as unknown species. The library clone with set A universal primers amplifying 350 bp displayed increased diversity by days. Thus, set A primers were better suited for this type of molecular ecological analysis. On the first day of the life of the infant, Enterobacter, Lactococcus lactis, Leuconostoc citreum, and Streptococcus mitis were present. The largest taxonomic group was L. lactis. On the third day of the life of the infant, Enterobacter, Enterococcus faecalis, Escherichia coli, S. mitis, and Streptococcus salivarius were present. On the sixth day of the life of the infant, Citrobacter, Clostridium difficile, Enterobacter sp., Enterobacter cloacae, and E. coli were present. The largest taxonomic group was E. coli. These results showed that microbiotic diversity changes very rapidly in the few days after birth, and the acquisition of unculturable bacteria expanded rapidly after the third day.