• Journal of Microbiology and Biotechnology
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• 제28권7호
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• pp.1141-1146
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• 2018

2'-Fucosyllactose (2'-FL) is one of the most important human milk oligosaccharides and has several health benefits for infants. The levels of 2'-FL in breast milk or samples from other sources can be quantified by high-performance liquid chromatography. However, this method cannot be used for simultaneous detection of the target compound in numerous samples. Here, we developed a simple method for quantifying 2'-FL in a microplate format. The method involves two steps: (i) release of $\text\tiny{L}$-fucose from 2'-FL by ${\alpha}$-(1-2,3,4,6)-$\text\tiny{L}$-fucosidase and (ii) measurement of NADPH formed during the oxidation of $\text\tiny{L}$-fucose by $\text\tiny{L}$-fucose dehydrogenase. This method enables measurement of up to 5 g/l 2'-FL in 50 min using a 96-well microplate. The efficiency and simplicity of the proposed method make it suitable for the analyses of a large number of samples simultaneously.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제22권4호
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• pp.330-340
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• 2019

Human breast milk contains numerous biomolecules. Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk, after lactose and lipids. Amongst the synthetized HMOs, 2'-fucosyllactose (2'-FL) and lacto-N-neotetraose (LNnT) are widely studied and are considered safe for infant nutrition. Several studies have reported the health benefits of HMOs, which include modulation of the intestinal microbiota, anti-adhesive effect against pathogens, modulation of the intestinal epithelial cell response, and development of the immune system. The amount and diversity of HMOs are determined by the genetic background of the mothers (HMO secretors or non-secretors). The non-secretor mothers secrete lower HMOs than secretor mothers. The breastfed infants of secretor mothers gain more health benefit than those of non-secretor mothers. In conclusion, supplementation of infant formula with 2'-FL and LNnT is a promising innovation for infant nutrition.

• Journal of Microbiology and Biotechnology
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• 제32권11호
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• pp.1471-1478
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• 2022

2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (Kan^R) should be dependent on FucT2 solubility. Kan^R was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.

• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.356-362
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• 2023

The 2'-fucosyllactose (2'-FL) is the richest components in a human milk oligosaccharide. Several studies have reported that 2'-FL has beneficial effects in infants. However, there are few studies on its immune-enhancing effects. This research aimed to examine the immune-enhancing effect of 2'-FL on immunosuppression by cyclophosphamide (CCP) in ICR mice. Mice were orally administered distilled water or 0.5 mg/kg B.W. 2'-FL for 14 days. An immunocompromised mouse model was induced using CCP 80 mg/kg B.W. at 12-14 days. Using the CCP had effects on reducing their body weight, organ weight, spleen index, natural killer (NK) cell activity, and cytokines concentration and expression. This study also used concanavalin A-mediated T-cell proliferation to verify the immune-enhancing effects in the sample. Body weight, spleen index, organ weight, and cytokine levels were measured to estimate the immune-enhancing effects. The body weight at 14 days tended to increase, and the spleen weight and index significantly increased in the 2'-FL group compared to the CCP group. The NK cell activity increased in the 2'-FL group compared to the CCP group, but there was no significant difference. The concentration of interleukin (IL)-2 tended to recover in the 2'-FL group compared to the CCP group. The 2'-FL group showed a significant increase of IL-10 and IFN-gamma concentration compared to the CCP group. In addition, there was a trend of increased IL-10 mRNA expression compared to the CCP group. These results revealed that 2'-FL improved CCP-induced immunosuppression, suggesting that 2'-FL may have the potential to enhance the immune system.

• 대한화장품학회지
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• 제48권2호
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• pp.105-112
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• 2022

2'-fucosyllactose (2'-FL)는 사람의 모유에 가장 많이 존재하는 올리고당(human milk oligosaccharides, HMOs)으로, 장내 유용 미생물의 성장을 촉진시키고 알레르기, 염증 반응을 완화시키는 것에 도움을 준다. 다양한 긍정적 기능을 가진 2'-FL의 미백 화장품 소재로써의 가능성을 확인하고자, 본 연구를 통해 멜라닌 생성 저해 효능 및 자가포식 유도 가능성을 검토하였다. 인간 유래 멜라닌 생성 세포로 알려진 MNT-1 세포에서 독성 실험을 진행하여 40 g/L 이하에서 세포독성이 없음을 확인하였고, 동일 세포에서 20 g/L 농도로 7 일간 처리하여 멜라닌 생성량을 분석한 결과, 40% 멜라닌 생성 감소를 확인하였다. 멜라닌 생성 관련 인자 TYR 및 TYRP1의 단백질 발현량을 western blot 법을 이용하여 분석한 결과, 2'-FL 처리는 이들을 감소시켰으며, 더불어 자가포식 표지자인 microtubule-associated protein 1 light chain 3 (LC3)의 형태가 LC3-I에서 LC3-𝚷로 변환을 확인할 수 있었다. 공초점 현미경을 통해 2'-FL 처리에 따른 LC3 puncta의 증가가 확인되었다. 따라서, 2'-FL로 활성화된 자가포식이 TYR 및 TYRP1 단백질 발현량을 저해시킴으로서 멜라닌 생성을 감소시키는 것으로 시사된다. 결론적으로 2'-FL은 자가포식을 유도하여 멜라닌 생성이 억제됨이 확인되어 미백 화장품 소재로써의 가능성이 기대된다.

• 한국식품영양학회지
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• 제36권3호
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• pp.193-201
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• 2023

Prebiotics are known as components of intestinal microbiota that can improve and maintain human health status by stimulating the growth and activity of the intestinal tract as a method of controlling the intestinal environment. In this study, we examined whether 2'-fucosyllactose (FL) could affect intestinal microbial population and bowel activity. Water content and frequency of mouse feces were increased in the 2'-FL treated group at a high concentration (1,000 mg/kg), with brightness of the color enhanced and physical properties diluted. In addition, intestinal microbial analysis showed that harmful bacteria Clostridium and Staphylococcus strains were decreased and beneficial bacteria such as Lactobacillus strains were markedly increased in the group treated with a high concentration of 2'-FL compared to those in the control group. These findings suggest that administration of 2'-FL can maintain healthy bowel activity by reducing harmful bacteria population and improving diluted physical properties.

• 한국식품과학회지
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• 제28권5호
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• pp.987-993
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• 1996

본 연구에서는 Bifidobacterium을 우유와 두유에 접종하여 $37^{\circ}C$에서 발효 특성, 적정 발효 기간 및 효율적인 증식을 위한 생육 인자를 찾고자 하였다. 이를 위하여 pH, 산도, glucose함량, 생균수, ${\alpha}-galactosidase$활성 그리고 ${\beta}-galactosidase$활성 변화를 조사하였다. $37^{\circ}C$에서 일반 발효 특성을 측정했을 때 48시간동안 우유의 발효는 pH의 감소와 산 생성의 증가는 현저한 반면, 세포 증식을 24시간동안 증가하였으나 남은 24시간동안은 감소를 보였다. 또한 효소 반응은 ${\beta}-galactosidase$ 활성이 높은 반면, ${\alpha}-galactosidase$는 비교적 낮았으며 glucose 함량은 세포 증식기에 높았다. 두유의 경우는 우유에 비해 pH의 감소가 큰 반면 적정산도 증가는 비슷하였다. 세포 증식은 발효 2일동안 이루어졌으며, ${\alpha}-,\;{\beta}-galactosidase$ 모두 활성이 높게 나타났고, glucose 함량도 1일 동안 지속적인 증가를 나타냈다. 또한 적정 발효기간은 발효유는 24-36시간, 발효 두유는 약 24시간이 바람직한 것으로 나타났다. Bifidobacterium의 성장 촉진 인자의 영향을 발효 시간에 따른 pH와 생균수 변화로 조사한 결과, 두유에서는 fucosyllactose, 그리고 우유에서는 발효 초기에 L-cysteine·HCl, 발효 진행중에는 fucosyllactose가 효과적인 생육 인자로 작용하였다.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제26권5호
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• pp.266-276
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• 2023

Purpose: Human milk oligosaccharides (HMOs) may be genetically determined based on the secretor and Lewis status of the mother. This study aims to determine the HMO profile and the secretor and Lewis gene status of Indonesian lactating mothers. Methods: Baseline data of 120 mother-infant pairs between 0-4 months post-partum obtained from a prospective longitudinal study was used. The concentrations of 2'-fucosyllactose (2'FL), lacto-N-fucopentaose I (LNFP I), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'SL), and 6'-sialyllactose (6'SL) were measured. Genetic analysis was performed for mothers using targeted next-generation sequencing and Sanger sequencing. Wild-type AA with the rs1047781 (A385T) polymorphism was categorized as secretor positive, while heterozygous mutant AT was classified as a weak secretor. The presence of rs28362459 (T59G) heterozygous mutant AC and rs3745635 (G508A) heterozygous mutant CT genes indicated a Lewis negative status, and the absence of these genes indicated a positive status. Subsequently, breast milk was classified into various groups, namely Group 1: Secretor+Lewis+ (Se+Le+), Group 2: Secretor-Lewis+ (Se-Le+), Group 3: Secretor+Lewis-(Se+Le-), and Group 4: Secretor-Lewis- (Se-Le-). Data were analyzed using the Mann-Whitney and Kruskal-Wallis rank tests, and a p-value of 0.05 indicated statistical significance. Results: A total of 58.3% and 41.7% of the samples had positive and weak secretor statuses, respectively. The proportion of those in Group 1 was 85%, while 15% were Group 3. The results showed that only 2'FL significantly differed according to the secretor status (p-value=0.018). Conclusion: All Indonesian lactating mothers in this study were secretor positive, and most of them had a Lewis-positive status.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제23권1호
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• pp.1-14
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• 2020

The latest definition of a prebiotic is "a substrate that is selectively utilized by host microorganisms conferring a health benefit"; it now includes non-food elements and is applicable to extra-intestinal tissues. Prebiotics are recognized as a promising tool in the promotion of general health and in the prevention and treatment of numerous juvenile diseases. Prebiotics are considered an immunoactive agent, with the potential for long-lasting effects extending past active administration of the prebiotic. Because of its extremely low risk of serious adverse effects, ease of administration, and strong potential for influencing the composition and function of the microbiota in the gut and beyond, the beneficial clinical applications of prebiotics are expanding. Prebiotics are the third largest component of human breast milk. Preparations including galactooligosaccharides (GOS), fructooligosaccharides (FOS), 2'-fucosyllactose, lacto-N-neo-tetraose are examples of commonly used and studied products for supplementation in baby formula. In particular, the GOS/FOS combination is the most studied. Maintaining a healthy microbiome is essential to promote homeostasis of the gut and other organs. With more than 1,000 different microbial species in the gut, it is likely more feasible to modify the gut microbiota through the use of certain prebiotic mixtures rather than supplementing with a particular probiotic strain. In this review, we discuss the latest clinical evidence regarding prebiotics and its role in gut immunity, allergy, infections, inflammation, and functional gastrointestinal disorders.