Detection of the mycotoxin citrinin using silver substrates and Raman spectroscopy

Singh, D.K.,Ganbold, E.O.,Cho, E.M.,Cho, K.H.,Kim, D.,Choo, J.,Kim, S.,Lee, C.M.,Yang, S.I.,Joo, S.W. Elsevier Scientific Pub. Co 2014 Journal of hazardous materials Vol.265 No.-

We detected a trace amount of the mycotoxin citrinin using surface-enhanced Raman scattering (SERS) on silver nanoparticle (Ag NP) surfaces. The SERS substrate on hydrophobic Teflon films was also introduced to observe the citrinin peaks. A broad band at ~1382cm<SUP>-1</SUP>, which was ascribed to the symmetric carboxylate stretching mode, was observed in addition to an antisymmetric carboxylate stretching mode at ~1568cm<SUP>-1</SUP> in the Raman spectra. The spectral feature indicated that citrinin would adsorb on Ag NPs via its carboxylate form. Based on density functional theory (DFT) calculations, vibrational mode analysis was performed to compare the Raman spectra of citrinin. DFT calculations also predicted that a bidentate bridge configuration through O15 and O16 atoms in citrinin would be the most stable on three Ag atoms. After treating with Ag NPs, observation of citrinin peaks was attempted in fungal cells of Penicillium citrinum. This work may provide useful insights into the direct observation of the hazardous citrinin mycotoxin using SERS by understanding its adsorption behaviors on Ag surfaces.

A Monascus pilosus MS-1 strain with high-yield monacolin K but no citrinin

Feng, Yanli,Chen, Wanping,Chen, Fusheng 한국식품과학회 2016 Food Science and Biotechnology Vol.25 No.4

Monacolin K (MK) produced by Monascus spp. is mostly used to regulate cholesterol level, while it can be contaminated by citrinin, a mycotoxin yielded by some Monascus strains. To develop production of citrinin-free MK, an M. sp. MS-1 strain, identified as M. pilosus by morphologies and internal transcribed spacer sequences analysis, was isolated from red fermented rice. After 14 days of fermentation, yields of citrinin-free MK could be up to 0.58 mg/mL and 16.45 mg/g in liquid- and solid-state fermentation products under optimal conditions, respectively. Further determination revealed that no citrinin biosynthesis related genes such as ctnA, pksCT, ctnE, and ctnR were detected. Thus, HPLC combined with citrinin-related gene analyses can be used for rapid screening of non-citrinin production Monascus strains.

Statistical Optimization for Monacolin K and Yellow Pigment Production and Citrinin Reduction by Monascus purpureus in Solid-State Fermentation

( Jirasatid Sani ),( Montira Nopharatana ),( Panit Kitsubun ),( Taweerat Vichitsoonthonkul ),( Anan Tongta ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.3

Monacolin K and yellow pigment, produced by Monascus sp., have each been proven to be beneficial compounds as antihypercholesterolemic and anti-inflammation agents, respectively. However, citrinin, a human toxic substance, was also synthesized in this fungus. In this research, solidstate fermentation of M. purpureus TISTR 3541 was optimized by statistical methodology to obtain a high production of monacolin K and yellow pigment along with a low level of citrinin. Fractional factorial design was applied in this study to identify the significant factors. Among the 13 variables, five parameters (i.e., glycerol, methionine, sodium nitrate, cultivation time, and temperature) influencing monacolin K, yellow pigment, and citrinin production were identified. A central composite design was further employed to investigate the optimum level of these five factors. The maximum production of monacolin K and yellow pigment of 5,900 mg/kg and 1,700 units/g, respectively, and the minimum citrinin concentration of 0.26 mg/kg were achieved in the medium containing 2% glycerol, 0.14% methionine, and 0.01% sodium nitrate at 25oC for 16 days of cultivation. The yields of monacolin K and yellow pigment were about 3 and 1.5 times higher than the basal medium, respectively, whereas citrinin was dramatically reduced by 36 times.

Monascus anka를 이용한 홍국주의 제조 및 특성

방병호 ( Byung Ho Bang ),이문수 ( Moon Soo Rhee ),김관필 ( Kwan Pil Kim ),이기원 ( Ki Won Lee ),이동희 ( Dong Heui Yi ) 한국식품영양학회 2013 韓國食品營養學會誌 Vol.26 No.1

홍국주의 제조 방법을 재현하고 그 기능성을 탐색하고자 Monascus anka KCTC 6121로 홍국을 제조, 이를 이용하여 홍국주를 제조하여 발효 과정 중의 pH, 총 산도, 환원당, 알코올 및 색소(황색, 적색, monacolin K 및 citrinin) 등의 성분 변화를 분석하였다. 홍국주의 pH는 1차 담금 직후에는 약간의 차이(pH 4.08∼4.58)를 나타냈으나, 배양 9일째 4구(H1, H2, H3 및 H4) 모두 비슷한 3.70 부근으로 나타났다. 총 산도는 배양 직후에는 별 차이가 없었으나(4구 모두 0.2% 부근), 배양 마지막 날인 9일째 홍국만으로 제조한 홍국주(H1)는 0.69%, 누룩을 첨가한 홍국주(H2∼H4)는 0.74∼0.76%의 값을 나타내었다. 잔존 환원당은 발효 마지막 날인 8일째 미미하지만, 누룩 첨가량이 많을수록 더 적게 나타났다. 누룩의 첨가량이 높을수록 당의 소비량은 높았으며, 이에 따라 알코올 농도는 12∼13.7%까지의 차이를 보였다. 발효 9일째, 황색 색소의 함량(7.2∼8.8 OD unit)은 적색 색소 함량(4.4∼5.1 OD unit)보다 높은 값을 나타났으며, 그리고 monacolin K와 citrinin의 함량은 각각 9.48㎎/㎏, 10.14㎎/㎏으로 검출되었다. 이상에서 살펴본 바와 같이 홍국을 술의 효소제로 사용하여 홍국주 제조를 이용한 결과, 술로서의 이용 가능성은 높았으나, citrinin의 함량이 높게 나와 홍국 제조 시 citrinin의 생성을 줄이고, 색소 및 효소 활성을, monacolin K의 생성을 높일 수 있도록 균주의 개량을 통해 홍국주의 기능성 및 기호성 면에서 보다 효율적으로 이용될 수 있는 추후 연구가 필요할 것으로 사료된다. To reproduce the brewing process of Hongkuk-ju and to identify the functional properties of it, Hongkuk-ju was brewed using different additions of Hongkuk (100%, 90%, 70%, 50%) and Nuruk (0%, 10%, 30%, 50%). The quality elements, including pH, total acidity, reducing sugar content, alcohol content and pigments (yellow, red, monacolin K and citrinin), were measured. The pH values of Hongkuk-ju showed a slight difference (pH 4.08∼4.58) right after the 1st stage mash; further, the pH on all groups (H1, H2, H3 and H4) in the terminal of the 2nd stage mash (9 days in fermentation) were similar, ranging approximately at pH 3.70. The total acidity change did not show a difference directly the 1st stage mash (nearby 0.2 %); however, it began to show a slight difference at the terminal of the 2nd stage mash between the range of 0.69∼0.76%. The residual reducing sugar of the content was decreased with the increased Nuruk content. The alcohol concentrations of the treatment brew with Nuruk ranging from 12.3% to 13.7% were higher than Hongkuk on its own. The yellow and red pigment contents of Hongkuk-ju ranged from 7.2∼8.8 O.D. units (yellow pigment) and from 4.4∼5.1 O.D. units (red pigment). The production of monacolin K and citrinin was the highest (9.48㎎/㎏ and 10.14㎎/㎏) when the treatment solely brewed Hongkuk. The concentration of Nuruk and the preparation of the seed mash from it were critical factors compared to the treatment of rice in brewing Hongkuk-ju.

A Monascus pilosus MS-1 strain with high-yield monacolin K but no citrinin

Yanli Feng,Wanping Chen,Fusheng Chen 한국식품과학회 2016 Food Science and Biotechnology Vol.25 No.4

Monacolin K (MK) produced by Monascus spp. is mostly used to regulate cholesterol level,while it can be contaminated by citrinin, a mycotoxin yielded by some Monascus strains. To developproduction of citrinin-free MK, an M. sp. MS-1 strain, identified as M. pilosus by morphologies andinternal transcribed spacer sequences analysis, was isolated from red fermented rice. After 14 days offermentation, yields of citrinin-free MK could be up to 0.58 mg/mL and 16.45 mg/g in liquid- and solidstatefermentation products under optimal conditions, respectively. Further determination revealedthat no citrinin biosynthesis related genes such as ctnA, pksCT, ctnE, and ctnR were detected. Thus,HPLC combined with citrinin-related gene analyses can be used for rapid screening of non-citrininproduction Monascus strains.

홍국균 발효가 발아현미의 Monacolin K 함량과 항산화 활성에 미치는 영향

이상훈(Sang Hoon Lee),장귀영(Gwi Yeong Jang),김민영(Min Young Kim),김신제(Shinje Kim),이연리(Yuon Ri Lee),이준수(Junsoo Lee),정헌상(Heon Sang Jeong) 한국식품영양과학회 2015 한국식품영양과학회지 Vol.44 No.8

발아 온도와 기간에 따른 발아현미 홍국배양물의 홍국색소, monacolin K, citrinin 및 페놀 화합물 생성량과 항산화 활성 변화를 조사하였다. 발아 기간이 증가함에 따라 명도는 증가하였으나 적색도와 황색도는 감소하였고, 적색(A500), 오렌지색(A470) 및 황색(A400) 색소 생성량도 동일한 경향이었다. 총 monacolin K는 현미(215.85 mg/kg)가 백미(40.41 mg/kg)보다 높았으며, 32°C에서 1일간 발아시킨 발아현미(1,263.04 mg/kg)에서 가장 높은 함량을 보였으나 발아 기간이 증가함에 따라 감소하였다. 곰팡이 독소인 citrinin은 모든 처리구에서 검출되지 않았다. 총 폴리페놀과 플라보노이드 함량은 발아 온도와 기간이 증가함에 따라 유의적으로 증가하여 37°C에서 4일간 발아시킨 발아현미에서 13.80 mg/g과 1.30 mg/g으로 가장 높았다. 전자공여능과 총 항산화력은 발아에 의해 증가되어 32°C에서 1일간 발아시킨 발아현미에서 각각 22.16 mg TE/g과 62.27 mg AAE/g으로 가장 높았으나 발아 기간이 증가함에 따라 감소하였다. 이상의 결과로부터 홍국색소와 monacolin K 생성량을 증가시키기 위한 기질로 발아현미가 유용하며, 배양에 사용된 Monascus pilosus 305-9는 citrinin을 생성하지 않고 monacolin K 생성량을 증가시켜 식품의 기능성 증진용 소재 및 균주로서의 활용이 기대된다. This study was performed to investigate the changes in monacolin K content and antioxidant activities of Monascus-fermented brown rice with different germination temperatures and periods. Brown rice was germinated at 32, 35 and 37°C for 1∼4 days, after white rice (WB), brown rice (BR), and germinated brown rice (GBR) were fermented with M. pilosus 305-9 at 30°C for 20 days. The redness, yellowness and Monascus pigments increased after germination. Total monacolin K content increased from 215.85 mg/kg of BR to 1,263.04 mg/kg of GBR (32°C/1 day), whereas monacolin K content decreased with increase in germination period. Citrinin was not detected in any of the samples. Total polyphenol (TPC) and flavonoid contents (TFC) increased with increase in germination temperature and period, whereas electron donating ability (EDA) and total antioxidant activities (TAA) decreased due to reduction of Monascus pigment content. The TPC and TFC showed the highest values (13.80 mg/g and 1.30 mg/g, respectively) in GBR (37°C/4 day), whereas EDA and TAA showed the highest values (22.16 mg Trolox equivalent/g and 62.27 mg ascorbic acid equivalent/g, respectively) in GBR (32°C/1 day). These results indicated that the optimal germination temperature and period for increasing monacolin K content and antioxidant activities was found to be at 32°C for 1 day. In addition, it was found that M. pilosus 305-9 was a useful strain for increasing monacolin K content without producing citrinin in functional foods and pharmaceutical industrial regions.

The effect of citrinin extracts in VEGF-mediated angiogenesis in dermal endothelial cell

( Yun Sun Moon ),( Young Bin Shin ),( Jeong Won Jo ),( Hae Bong Jeong ),( Do Seon Jeong ),( Sang Min Lee ),( Eui Chang Jung ),( Chi Yeon Kim ),( Tae Jin Yoon ) 대한피부과학회 2017 대한피부과학회 학술발표대회집 Vol.69 No.2

Background: Cutaneous carcinogenesis is a multistep process in which clinically recognizable pre-stages occasionally occur. The importance of angiogenesis in tumor progression has been highlighted by studies showing that the angiogenic potential of tumors developed skin. Objectives: In the present study, we investigated the antiangiogenic activities of citrinin present in red yeast rice, widely used in Asia as a food additive in both in skin tumor and in vitro study with human vascular endothelial cell. Methods: We assessed angiogenesis in skin epithelial cell tumor specimens(64 specimens from 54 patients) in progressing step actinic keratosis, squamous cell carcinoma in situ, and squamous cell carcinoma by evaluated with microvessel density(MVD) and the expression of vascular endothelial growth factor(VEGF) . We evaluated the effect of citrinin to angiogenic activity to human vascular endothelial cell treated to angiogenic promoter VEGF. Results: The vascular density and expression of VEGF more increased squamous cell carcinoma than actinic keratosis in full thickness of epithelial layer and dermal vascular structure. The result presented that citrinin has antiangiogenic effect to VEGF induced angiogenesis in dose dependent manner might be the prevalent and immediate therapeutic mechanism of the progression of tumorigenesis. Conclusion: This study provides useful data on the topical therapeutic modality include citrinin to precancerous lesion progress to skin cancer.

The Possible Mechanisms Involved in Citrinin Elimination by Cryptococcus podzolicus Y3 and the Effects of Extrinsic Factors on the Degradation of Citrinin

( Xiaoyun Zhang ),( Zhen Lin ),( Maurice Tibiru Apaliya ),( Xiangyu Gu ),( Xiangfeng Zheng ),( Lina Zhao ),( Mandour Haydar Abdelhai ),( Hongyin Zhang ),( Weicheng Hu ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.12

Citrinin (CIT) is a toxic secondary metabolite produced by fungi belonging to the Penicillium, Aspergillus, and Monascus spp. This toxin has been detected in many agricultural products. In this study, a strain Y3 with the ability to eliminate CIT was screened and identified as Cryptococcus podzolicus, based on the sequence analysis of the internal transcribed spacer region. Neither uptake of CIT by cells nor adsorption by cell wall was involved in CIT elimination by Cryptococcus podzolicus Y3. The extracellular metabolites of Cryptococcus podzolicus Y3 stimulated by CIT or not showed no degradation for CIT. It indicated that CIT elimination was attributed to the degradation of intracellular enzyme(s). The degradation of CIT by C. podzolicus Y3 was dependent on the type of media, yeast concentration, temperature, pH, and initial concentration of CIT. Most of the CIT was degraded by C. podzolicus Y3 in NYDB medium at 42 h but not in PDB medium. The degradation rate of CIT was the highest (94%) when the concentration of C. podzolicus Y3 was 1 × 10⁸ cells/ml. The quantity of CIT degradation was highest at 28°C, and there was no degradation observed at 35°C. The study also showed that acidic condition (pH 4.0) was the most favorable for CIT degradation by C. podzolicus Y3. The degradation rate of CIT increased to 98% as the concentration of CIT was increased to 20 μg/ml. The toxicity of CIT degradation product(s) toward HEK293 was much lower than that of CIT.

Citrinin Hydrate Inhibits Serotonin N-Acetyltransferase Catalyzing the Conversion of Serotonin to N-Acetylserotonin

LEE, IN-KYOUNG,YUN, BONG-SIK,KIM, KYONG-TAI,CHOI, BO-HWA,PARK, TAE-JU,KIM, YOUNG-HO,YOO, ICK-DONG 한국미생물 · 생명공학회 2001 Journal of microbiology and biotechnology Vol.11 No.6

In an attempt to search for serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT) inhibitors from microbial metabolites, we found the culture broth of Penicillium sp. 80722 which showed a strong inhibitory activity against AA-NAT. The active principle has been identified as citrinin hydrate through bioassay-guided fractionation of cultural broth, and structure elucidation derived by spectroscopic analyses. Citrinin hydrate inhibits AA-NAT with an IC_50 value of 173μM in a dose-dependent manner. Although citrinin hydrate was previously isolated as human rhinovirus 3C-protease inhibitor, this was recognized as the first AA-NAT inhibitor isolated from natural sources.

Optimal Selection of Agricultural Products to Inhibit Citrinin Production during Submerged Culture of Monascus anka

Xu Xiong,Xuehong Zhang,Zhenqiang Wu,Zhilong Wang 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.6

Agricultural products exhibit a “buffering nature”during autoclaving and fermentation processes: i.e., thefinal pH of agricultural product medium after Monascusfermentation maintains a certain value. The protein contentof agricultural products strongly affects the bufferingcapacity and an extremely low final pH, below 3, can beachieved by the selection of agricultural products with lowprotein content, such as rice meal and cornmeal. This lowpH can be used to inhibit citrinin biosynthesis duringMonascus fermentation. Thus, optimal selection of agriculturalproducts with low protein content may provide a novelstrategy to inhibit citrinin production in submerged cultureof Monascus anka at a low initial pH for the production ofMonascus pigments. pH values ranging from 2.6 to 3 canbe maintained in Monascus fermentation using cornmeal/wheat starch (30:30 g/L) as substrate at initial pH 2.5,where the concentration of intracellular pigments reaches45 AU at 470 nm while citrinin is undetectable by TLCanalysis.