Bacillus subtilis 유래 재조합 endoxylanase를 이용한 xylooligosaccharide의 최적 생산

김연희(Yeon-Hee Kim),허선연(Sun-Yeon Heo),김미진(Mi-Jin Kim),이재형(Jae Hyung Lee),김영만(Young-Man Kim),남수완(Soo-Wan Nam) 한국생명과학회 2008 생명과학회지 Vol.18 No.1

식물체 구성성분의 하나인 hemicellulose의 대부분을 차지하는 xylan은 농산 폐기물 또는 목재 성분의 약 30%를 차지하는 풍부한 자원물질이다. 이러한 xylan을 효과적으로 분해하기 위해 Bacillus에서 발현시킨 재조합 endoxylanase를 이용하여 기능성 식품소재인 xylooligosaccharide의 최적 생산 조건을 조사하였다. B. subtilis 재조합 균주 DB431/pJHKJ4를 이용한 발효조 회분배양 결과, endoxylanse의 총활성은 857 unit/ml이며, 대부분이 세포밖으로 분비됨을 알 수 있었고, 분비효율은 92%로 나타났다. 재조합 endoxylanase를 이용하여 xylan으로부터 xylooligosaccharide 생성을 위한 최적 반응조건을 검토한 결과, xylooligosaccharide 생산을 위한 기질로는 birchwood xylan이 적합함을 알 수 있었고, 4%의 xylan 농도에서 가장 많은 xylooligosaccharide을 생산하며, xylobiose와 xylotriose가 주생성물임을 알았다. 효소의 농도와 반응시간의 영향은 10 unit의 endoxylanase를 첨가하여 1시간 반응시켰을 때 가장 많은 양의 xylooligosaccharide을 생산할 수 있었고, 반응온도는 40~50℃가 적합함을 알았다. 결론적으로 재조합 endoxylanase을 이용한 xylooligosaccharide생성에는 10 unit endoxylanase과 4% birchwood xylan을 기질로 이용하여 50℃에서 1시간 반응시키는 것이 최적반응 조건임을 알았다. Xylan is a major hemicellulose component of the cell walls of monocots and hardwood, representing up to 30% of the dry weight of these plants. To efficiently hydrolyze xylan, the endoxylanase gene from Bacillus sp. was expressed in B. subtilis DB431 by introducing the plasmid pJHKJ4. The total activity of the recombinant endoxylanase reached about 857 unit/ml by batch fermentation of B. subtilis DB431/pJHKJ4 in LB maltose medium. The majority (>92%) of endoxylanase was efficiently secreted into the culture medium. The recombinant endoxylanase hydrolyzed more the birchwood xylan efficiently than the other xylans. When 4% concentration of xylan was used, the highest production of xylooligosaccharide was observed, and xylobiose and xylotriose were the major products. Optimal amount of enzyme and reaction time for producing xylooligosaccharide were found to be 10 unit and 1 hr, respectively. In addition, the temperature of 40℃~50℃ gave the highest production of xylooligosaccharide. Consequently, the optimized conditions for the production of xylooligosaccharide through the hydrolysis of xylan were determined as follows: 10 unit endoxylanase, 50℃, 4% birch-wood xylan, 1 hr reaction.

자일로올리고당을 함유한 설탕이 20대 여성의 변비 개선에 미치는 효과: 이중맹검 연구

전진호,경명옥,정상원,조성은,장문정 한국영양학회 2015 Journal of Nutrition and Health Vol.48 No.1

Purpose: To investigate the effects of the intake of xylooligosaccharide-sugar mixture (XOS) on defecation frequency andsymptoms in 56 young women (mean age of 22.1 years old) with constipation. Methods: Two experiments were conducted. In experiment 1, a randomized double-blind study was performed to evaluate the effect of 6 weeks' intake of 10 g sucrosecontaining 7% xylooligosaccharide or 10 g sucrose on constipation. In experiment 2, 24 g coffee mixture containing 12.8 gplant cream and 11.2 g xylooligosaccharide-sugar mixture was consumed by the subjects. During the study, the clinicalefficacy was assessed by using a daily diary. The subjects indicated the number of frequencies they defecated in a dayand the clinical symptom scores. Results: In experiment 1, the mean frequency of defecations was 2.07 in the pretreatmentweek and increased significantly to 4.05, 4.42, 4.84, 4.84, and 4.05 in weeks 2 to 6 of XOS intake, in comparison with the3-3.67 with sucrose intake (sucrose, SUC). In experiment 2, the mean frequency of defecations significantly increased from2.47 in the pretreatment week to 4.11-5.67 in weeks 1-6 of XOS intake. The occurrence of very loose or loose stools in theXOS group was significantly increased in weeks 5 and 6, compared with the pretreatment week and SUC group. XOS intakesignificantly alleviated the abdominal displeasure and feeling of residual stool leftness in weeks 2, 3, 5, and 6, while SUCdid so in weeks 4 and 6 (p < 0.05). The coffee mixture containing xylooligosaccharide-sugar mixture reduced theabdominal displeasure and feeling of residual stool leftness from week 3 until the end of the experiment (p < 0.05). Conclusion: Our results suggest that xylooligosaccharide-sugar mixture intake was effective, without adverse effects, forthe alleviation of constipation in the young women in this study. 본 연구는 자일로올리고당을 설탕에 혼합하여 변비를앓고 있는 젊은 여성을 대상으로 변비개선효과를 확인하였다. 평균연령 22.1세인 젊은 여성 56명을 대상으로 실험1에서는 설탕을 플라시보로 하는 이중맹검 임상연구를 통해 자일로올리고당 0.7 g이 함유된 설탕 10 g (n = 19명) 또는 설탕 10 g (n = 19명)을 블랙커피와 함께 복용하도록 하였고, 실험 2에서는 자일로올리고당 0.4 g 함유한 설탕 5.6 g과 1.6 g의 커피, 4.8 g의 식물성 크림이 함유된 커피믹스를 1일 2회씩 6주간 섭취하도록 하였다 (n = 18명). 섭취결과 XOS군과 XOSmix군의 경우 섭취전 주간 배변횟수가2.47회에서 섭취 1주째부터 증가하여 섭취 6주째에는 주간배변횟수가 4.11회와 5.67회로 각각 증가하였다. XOS와XOS 함유 커피믹스는 통계적으로 유의하게 복부 불쾌감과 잔변감을 개선하였으며, 배변횟수의 증가는 섭취초반부터 효과가 있는 것으로 나타났다. 따라서 XOS는 0.7 g의적은양으로도 변비를 개선하는 효과가 있는 것으로 나타나 음료에 기능성을 갖춘 감미료로서의 활용이 기대된다.

건강기능식품에서 HPLC-UVD를 이용한 자일로올리고당 시험법의 표준화 연구

이세윤,정희선,김규헌,이미영,최정호,안정선,권광일,이혜영,Se-Yun Lee,Hee-Sun Jeong,Kyu-Heon Kim,Mi-Young Lee,Jung-Ho Choi,Jeong-Sun Ahn,Kwang-Il Kwon,Hye-Young Lee 한국식품위생안전성학회 2024 한국식품위생안전성학회지 Vol.39 No.2

본 연구는 자일로올리고당의 과학적이고 체계적인 표준화된 시험법을 마련하여 다양한 제형의 제품에 적용하고자 하였다. 최적화된 시험법을 마련하기 위해 초음파 처리 시간, 산 가수분해 시간 및 농도를 검토하여 전처리 방법을 비교 평가하였으며, HPLC-UVD를 이용하여 시료 중의 자일로올리고당을 분석하였다. 분석 시 1-phenyl-3-methyl-5-pyrazoline (PMP)으로 유도체화하고, photo diode array (PDA)가 장착된 high performance liquid chromatography (HPLC) (Nanospace SI-2)를 사용하였으며, 칼럼은 Cadenza C₁₈ (4.6 × 250 mm, 3 ㎛)이었으며, 이동상은 pH를 6.0으로 맞춘 20 mM 인산완충용액과 아세토니트릴을 78:22 비율로 혼합하여 사용하였고, 0.5 mL/min 유속으로 254 nm로 하여 분석하였다. 건강기능식품 등 시험법 마련 표준절차에 관한 가이드라인에 따라 밸리데이션을 수행하고, 표준화된 시험법을 이용하여 유통 중인 건강기능식품 대상 품목에 대해 시험법 적용 여부를 확인하였다. 표준화된 시험법은 자일로올리고당을 함유한 건강기능식품 품질관리에 대한 신뢰성을 더 높일 것으로 본다. This study aimed to develop a scientifically and systematically standardized xylooligosaccharide analytical method that can be applied to products with various formulations. The analysis method was conducted using HPLC with Cadenza C₁₈ column, involving pre-column derivatization with 1-phenyl-3-methyl-5-pyrazoline (PMP) and UV detection at 254 nm. The xylooligosaccharide content was analyzed by converting xylooligosaccharide into xylose through acid hydrolysis. The pre-treated methods were compared and evaluated by varying sonication time, acid hydrolysis time, and concentration. Optimal equipment conditions were achieved with a mobile phase consisting of 20 mM potassium phosphate buffer (pH 6)-acetonitrile (78:22, v/v) through isocratic elution at a flow rate of 0.5 mL/min (254 nm). Furthermore, we validated the advanced standardized analysis method to support the suitability of the proposed analytical procedure such as specificity, linearity, detection limits (LOD), quantitative limits (LOQ), accuracy, and precision. The standardized analysis method is now in use for monitoring relevant health-functional food products available in the market. Our results have demonstrated that the standardized analysis method is expected to enhance the reliability of quality control for healthy functional foods containing xylooligosaccharide.

식품소재로서의 Xylooligosaccharide의 물리화학적 특성

박윤제(Youn-Je Park),이지완(Ji-Wan Lee),이창승(Chang-Seung Lee),류보경(Bo-Kyung Rhew),양창근(Chang-Kun Yang) 한국식품과학회 2001 한국식품과학회지 Vol.33 No.1

자일로올리고당을 함유한 설탕이 건강한 성인의 Glycemic Index (GI)와 혈당에 미치는 효과

경명옥,최한샘,정상원,이경선,조성은,서승우,최근범,양창근,유상호,김유리 한국영양학회 2014 Journal of Nutrition and Health Vol.47 No.4

Purpose: The objective of this study was to investigate the effects of xyloologosaccharide (XOS)-sugar mixture on glyce-mic index (GI) and blood glucose in human subjects. Methods: Randomized double-blind cross-over studies were con-ducted to examine the effect of sucrose with 14% xyloologosaccharide powder (Xylo 14) and sucrose with 20% xylooligo-saccharide powder (Xylo 20) on GI and postprandial glucose response at 15, 30, 45, 60, 90, and 120 min. Results: GIs of Xylo 14 and Xylo 20 were 60.0 ± 23.5 classified within medium GI range, and 54.3 ± 17.7 within low GI range, respectively. Xylo 14 and Xylo 20 showed significantly lower area under the glucose curve (AUC) for 0-15 min (p = 0.0113), 0-30 min (p = 0.0004), 0-45 min (p < 0.0001), 0-60 min (p < 0.0001), 0-90 min (p < 0.0001), and 0-120 min (p = 0.0001). In particular, compared with glucose, the blood glucose levels of Xylo 14 and Xylo 20 were significantly lower at every time point between 15 and 120 min. Conclusion: The results of this study suggested that Xylo 14 and Xylo 20 had an acute suppressive effect on GI and the postprandial glucose surge. 본 연구는 자일로올리고당을 설탕에 적용하여 임상시험을통해 자일로올리고당 첨가량에 따른 설탕의 GI 저감효과와 혈당저감효과를 확인하였다. 건강한 성인 남녀 11명를 대상으로표준식품인 포도당과 비교하여 분말 자일로올리고당을 14% 함유한 Xylo 14과 분말 자일로올리고당을 20% 함유한 Xylo 20을 섭취 후 표준식품과 비교식품의 IAUC를 통해 GI를 산출한 결과 Xylo 14는 60.0 ± 23.5로 Medium GI 식품으로 나타났으며, Xylo 20은 54.3 ± 17.7로 Low GI 식품으로 나타났다. 순수한 설탕의 GI 68과 비교하면 각각 11.8%와 20.1% 혈당상승 저감효과를 나타낼 수 있었다. AUC는 15~90분까지 모든 구간에서 유의적인 차이 (p < 0.05)를 나타내었다. 따라서Xylo 14와 Xylo 20은 혈당 저감효과를 나타냄과 동시에 7~10 g 이상 섭취하면 자일로올리고당의 1일 유효섭취량도 만족시킬 수 있으므로 기능성 설탕으로서의 활용이 기대된다.

제주 연안해수로부터 한천 분해 효소 및 자일란 분해 효소를 생산하는 Catenovulum jejuensis A28-5의 동정 및 특성 규명

김다솜 ( Da Som Kim ),정가람 ( Ga Ram Jeong ),배창환 ( Chang Hwan Bae ),여주홍 ( Joo-hong Yeo ),지원재 ( Won-jae Chi ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 한국미생물·생명공학회지 Vol.45 No.2

Strain A28-5는 대한민국 제주도 연안의 해수샘플로부터 고체배지내 xylan과 agar를 분해하는 균주로 분리되었다. Strain A28-5는 그람 음성균으로 한 개의 polar flagella로 운동성을 갖는 Na⁺ 이온 요구성 균주로 분석되었다. 또한 ampilcillin과 thiostreptone등의 항생제에 내성을 보였다. Genome 내 G+C content는 43.96%이고, Menaquinone-7 (MK-7)을 predominant quinone으로 함유하고 있었다. Strain A28-5의 세포벽을 구성하는 주요 지방산은 C_16:1 ω7c/iso-C_15:0 2-OH (23.32%), C_16:0(21.83%), C_18:1 ω7c(17.98%)였다. strain A28-5의 16S rRNA gene sequence는 Catenovulum agarivorans YM01와 가장 높은 상동성(98.94%)을 보였으며, Neighbor-Joining phylogenetic tree 제작을 통해서 Catenovulum agarivorans YM01와 가장 높은 근연관계를 보이는것을 증명하였다. Catenovulum agarivorans YM01과의 DNA-DNA hybridization 분석을 통하여 A28-5을 Catenovulum 속의 신종으로 분류하여Catenovulum jejuensis A28-5로 명명하였다. 이 균주의 액체배양으로부터 준비된 두 종류의 조효소를 이용한 xylan 또는 agarose 와의 효소반응액을 Thin layer chromatography로 분석하여 각각 tetramer와 hexamer의 xylooligosaccharides와 (neo)agarooligosacchardes가 생산되는 것을 확인하였다. Strain A28-5, which can degrade xylan and agar in solid medium, was isolated from a coastal seawater sample collected from Jeju Island, South Korea. This strain was found to be a gram-negative, Na⁺-requiring bacte-rial strain with a polar flagellum for motility. Additionally, the strain was tolerant to antibiotics such as ampicillin and thiostrepton. The G+C content of the genome was 43.96% and menaquinone-7 was found to be the predominant quinone. Major fatty acids constituting the cell wall of the strain were C_16:1 ω7c/iso-C_15:0 2-OH (23.32%), C_16:0 (21.83%), and C_18:1 ω7c (17.98%). The 16S rRNA gene sequence of the strain showed the highest similarity (98.94%) to that of Catenovulum agarivorans YM01, which was demonstrated by construct-ing a neighbor-joining phylogenetic tree. A28-5 was identified as a novel species of the genus Catenovulum via DNA-DNA hybridization with Catenovulum agarivorans YM01, and thus was named as Catenovulum jejuensis A28-5. The formation of tetramers and hexamers of xylooligosaccharides and (neo)agarooligosac-charides, respectively, were confirmed by thin-layer chromatography analysis using an enzyme reaction solution containing xylan or agarose with two crude enzymes prepared from the liquid culture of the strain.

자일로올리고당의 섭취가 변 내 비피더스 균수, Lactic Acid 농도와 지질대사에 미치는 영향

나미희(Na Mi Hee),김우경(Kim Woo Kyoung) 韓國營養學會 2007 Journal of Nutrition and Health Vol.40 No.2

This study investigated the effects of xylooligosaccharide on feces bifidobacteria proliferation, lactic acid concentration and lipid metabolism in healthy woman. Fourteen volunteers were randomly assigned to 2 groups : 1.4 g/day xylooligosaccharide intake group, 2.8 g/day xylooligosaccharide intake group. The duration of the study was 28 days. The amount of feces and excretion time were not affected by xylooligosaccharide intake. The color of feces changed to yellow brown, and hardness of stool and effort to evacuation were reduced by xylooligosaccharide intake. Xylooligosaccharide intake reduced the fecal pH significantly after 14 days in 2.8 g/day intake group (p<0.05). The number of fecal bifidobacteria were significantly increased after 28 days in 1.4 g/day intake group (p<0.05), and in 2.8 g/day intake group, the number of fecal bifidobacteria significantly increased after 14 days (p<0.05). Water contents of feces were not affected by xyloolgosacchride intake. The fecal triglyceride and cholesterol concentrations were increased in 2.8 g/day intake group (p<0.05), and in 1.4 g/day intake group, fecal cholesterol concentration only was increased (p<0.05). The fecal lactic acid concentration was significantly increased in 2.8 g/day intake group (p<0.05). Serum trigly-ceride, cholesterol and glucose concentration were significantly decreased in 2.8 g/day intake group (p<0.05). In conclusion, xylooligosaccharide dietary supplementation may be beneficial to gastrointestinal health and lipid metabolism, and 2.8 g/day intake was more effective than 1.4 g/day intake. (Korean J Nutrition 40(2): 154~161, 2007)

자일로올리고당 함유비율이 다른 설탕이 건강한 성인의 혈당지수와 혈당반응에 미치는 영향

남혜경(Nam, Hyekyoung),경명옥(Kyung, Myungok),서승우(Seo, Sheungwoo),정상원(Jung, Sangwon),장문정(Chang, Moon-Jeong) 한국영양학회 2015 Journal of Nutrition and Health Vol.48 No.5

본 연구는 8주 동안 건강한 성인남녀를 대상으로 설탕과 자일로 올리고당 함유비율이 다른 설탕 2종의 혈당 반응을 통한 GI를 비교하여 혈당 저하 효과를 확인하였다. 설탕의 GI는 68.9로 기존의 선행연구에서의 GI와 유사하였다. Xylo 7 (Xylooligosaccaride X2~X7 7% 함유), Xylo 10(Xylooligosaccaride X2~X7 10% 함유)은 포도당이나 설탕에 비해 섭취 후 최대 혈당 상승량이 낮았으며, Xylo 7과 Xylo 10은 통계적으로 유의하게 최대 혈당 상승량이 적었다. Xylo 7와 Xylo 10의 GI는 각각 54.7, 52.5으로 모두 저GI식품에 해당하여 일반 설탕과 비교하여 혈당상승을 유의하게 억제하였다. 또한 체지방률이 높을수록 Xylo 10의 섭취는 GI를 더 낮추는 경향이었다. 따라서 자일로올리고당 유효성분 X2~X7을 함유한 설탕은 혈당상승을 억제하는 건강 기능성을 갖춘 것으로 사료된다. Purpose: In the present study, we aimed to evaluate the effect of sucrose containing 2 different levels of xylooligosaccharide on the glycemic index (GI) and blood glucose response in healthy adults. Methods: Healthy adults (4 male participants and 6 female participants, n = 10) were randomized to receive glucose, sucrose, sucrose containing 7% xylooligosaccharide active elements (Xylo 7), or sucrose containing 10% xylooligosaccharide active elements (Xylo 10). Each participant was administrated one of these materials once a week for 8 weeks and an oral glucose tolerance test was performed. Results: We found a reduction in the glycemic response to sucrose that included xylooligosaccharide active elements (Xylo 7 and Xylo 10). The glycemic indices of sucrose, Xylo 7 and Xylo 10 were 68.9, 54.7, and 52.5, respectively. The GI values of Xylo 7 and Xylo 10 were similar to that of foods with low GI. The percentage reduction of GI value caused by sucrose containing xylooligosaccharide active elements was significantly different and dose-dependent as compared to that caused by sucrose alone (p < 0.05). The reduction in the glycemic response to Xylo 7 and Xylo 10 was 21% and 24%, respectively, as compared to the glycemic response to sucrose. The attenuation of the glycemic response to Xylo 10 tended to be higher than that for Xylo 7 when the percentage of body fat was increased. Conclusion: These results demonstrated that xylooligosaccharide active elements may be effective in protecting humans against overconsumption of sucrose.

Prebiotic Potential of Xylooligosaccharides Derived from Corn Cobs and Their In Vitro Antioxidant Activity When Combined with Lactobacillus

( Xiuhua Yu ),( Jianyuan Yin ),( Lin Li ),( Chang Luan ),( Jian Zhang ),( Chunfang Zhao ),( Shengyu Li ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.7

In the present work, the in vitro prebiotic activity of xylooligosaccharides (XOS) derived from corn cobs combined with Lactobacillus plantarum, a probiotic microorganism, was determined.These probiotics exhibited different growth characteristics depending on strain specificity. L. plantarum S2 cells were denser and their growth rates were higher when cultured on XOS. Acetate was found to be the major short-chain fatty acid produced as the end-product of fermentation, and its amount varied from 1.50 to 1.78 mg/ml. The antimicrobial activity of XOS combined with L. plantarum S2 was determined against gastrointestinal pathogens. The results showed that XOS proved to be an effective substrate, enhancing antimicrobial activity for L. plantarum S2. In vivo evaluation of the influence of XOS and L. plantarum S2, used both alone and together, on the intestinal microbiota in a mouse model showed that XOS combined with L. plantarum S2 could increase the viable lactobacilli and bifidobacteria in mice feces and decrease the viable Enterococcus, Enterobacter, and Clostridia spp. Furthermore, in the in vitro antioxidant assay, XOS combined with L. plantarum S2 possessed significant 2,2-diphenyl-1- picrylhydrazyl, 2,2’-azino-bis, and superoxide anion radical-scavenging activities, and the combinations showed better antioxidant activity than either XOS or L. plantarum S2 alone.

Production of Xylooligosaccharides from Corncobs Using Ultrasound-assisted Enzymatic Hydrolysis

Juntao Sun,Zhichao Zhang,Fugang Xiao,Ximei Jin 한국식품과학회 2015 Food Science and Biotechnology Vol.24 No.6

Xylooligosaccharides were obtained using ultrasound-assisted enzymatic hydrolysis from xylan that was alkali extracted from corncobs. Effects of alkali treatments on xylan extraction were investigated. The influence of operational conditions in ultrasound-assisted enzymatic hydrolysis on xylooligosaccharide production was also studied. Optimal conditions for xylan extraction were determined to be an alkaline concentration of 15%, a corncobs to alkaline solution ratio of 1:20, a reaction temperature of 100°C, and a reaction time of 2 h. Optimal conditions for xylanase-catalyzed hydrolysis of xylan to xylooligosaccharides were determined to be an enzyme concentration of 4 g/L, an ultrasound power of 200 W, a reaction temperature of 50°C and a reaction time of 10 min. Using a combination of ultrasound and enzyme increased the reducing sugar content by approximately 50%, compared with using of enzymes alone. Ultrasound and enzymes exert a synergistic effect.