Intergeneric Protoplast Fusion between Rhizopus oryzae and Aspergillus oryzae

Lee, Soo-Youn,Jung, Sung-Won,Kim, Seong-Han,Lee, Yung-Nok The Microbiological Society of Korea 1993 미생물학회지 Vol.31 No.3

Conditions for the release and regeneration of protoplasts form Rhizopus oryzae and intergeneric protoplast fusion between Rhizopus oryzae and Aspergillus oryzae were studied. High yields of protoplast fusion between Rhizopus oryzae and Aspergillus oxyzae were studied. High yield of protoplasts from young germilings of R. oryzae were obtained by using lytic enzymes containing chitosanase (3 mg/ml), chitinase (3 mg/ml) and Novozym 234 (5 mg/ml). 0.5M glucose was used as the osmotic stabilizer and optimum pH of buffer was determined to be pH 7.5-8.0. Under these conditions, protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts regenerated on solid medium with a soft agar overlay. We have also carried out protoplasts fusion between R. oryzae and A. oryzae and have succeeded in obtaining three types of intergeneric fusants. In these experiments, 35% PEG-4000 and 10 mM CaCl$_{2}$ were used as fsogenic agents, and auxotrophic properties were used as a genetic marker to select fusants. Complementation frequency be protoplasts fusion of A. oxyzae and R. oryzae was 4.4% * 10$^{-5}$ . The fusant strains of the first type were prototrophs showing an Aspergillus type morphology with dark-yellow sporulation, those of the second type were also Apergillus type morphology but showed no sporulation. And the strains of the third type stopped growing when fusion products grown on regeneration minimal medium were transferred to fresh minimal medium. The formation of fusion products was observed by fluorescent vital stains for complementary labelling of protoplats from R. oryzae and A. oryzae. Rhodamine 6G and fluorescein diacetate wer useful complementary vital stains of Rhizopus and Aspergillus protoplasts for visualization of requency and type (dicell, multicell) of fusion.

Aspergillus oryzae로 발효한 자색당근을 첨가한 발효유의 품질 및 관능 특성에 관한 연구

신배근,강선아,한정인,박선민 한국식생활문화학회 2015 韓國食生活文化學會誌 Vol.30 No.3

In this study, we compared the organoleptic and other qualities of fermented milk containing 10 or 15% purple carrot extract that had either been previously fermented with Aspergillus oryzae or not fermented. Fermentation characteristics, pH, chromaticity, viscosity, viable cell counts, and sensory evaluations were measured. The pH and acid values did not differ between purple carrot extract fermented with Aspergillus oryzae and non-fermented extract. Viable cell counts were significantly higher in 15% purple carrot extract fermented with Aspergillus oryzae compared to the control after fermentation. Regarding characteristic changes, purple carrot extract fermented with Aspergillus oryzae group showed a lower red value but higher yellow value compared with non-fermented purple carrot extract due to heat-sterilization. Both fermented and non-fermented extract groups showed significantly increased viscosity compared to control. In the sensory evaluation, 15% purple carrot extract fermented with Aspergillus oryzae showed the highest score. In conclusion, addition of 15% purple carrot extract fermented with Aspergillus oryzae resulted in a superior fermented milk product.

핵전이에 의한 Aspergillus oryzae var. oryzae와 Aspergillus nidulans 514의 잡종으로 부터 생산된 Xylanase의 특성

양영기,문명님,박형남,임채영 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.1

Aspergillus oryzae var oryzae 와 Aspergillus nidulans 514간의 xylanase 우수 생성 종간 형질전환체를 획득하고, 유전분석과 정제를 통하여 다음과 같은 결과를 얻었다. 1. 핵전이를 위한 원형질체 형성 및 재생 조건에서는 Novozym 234의 농도 1%, 삼투안정제는 0.6M (NH_4)_2 SO_4, 효소의 처리 시간은 90분 그리고 최적 pH는 5.8로 나타났다. 2. 핵전이에 의한 형질전환의 빈도는 3×10 exp (-5)~1×10 exp (-5)으로 비교적 낮은 편이었다. 3. 유전적 안정성, conidia의 크기, DNA 함량의 측정 그리고 핵염색의 결과 형질전환체의 핵형은 aneuploid로 추정되었다. 4. 형질전환체의 xylanase 활성은 모균주와 비교하여 1.1~1.4배 증가하였다. 5. 형질전환체중 xylanase 활성이 가장 높은 TAVD514-3을 정제하고 몇가지 특성을 조사하였는데 전체적인 회수율이 17%, 분자량은 32,000 그리고 효소 활성에 미치는 기질의 영향을 올 조사하였다. 6. 정제된 효소의 최적 pH와 온도는 각각 pH 9.0과 80℃로 나타났으며, 효소활성은 Mg^2+ 와 Mn^2+의 존재시에 증가하는 경향을 나타내었다. Inerspecific hybrids between Aspergillus oryzae var oryzae and A. nidulans 514 were obtained by nuclear transfer technique. Several auxotrophic mutants isolated from conidiospores of the two strains were mutagenized with ultraviolet and N-methyl-N-nitrosoguanidine. Optimal conditions for formation of intergeneric hybrids were investigated. Frequencies of hybrid formation by nuclear transfer were 3×10 exp (-5)~1×10 exp (-5). From observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyptypes are aneuploid. The hybrids showed 1.1~1.4 fold higher xylanase activities than parental strains did. The xylanase of Aspergiilus sp. TAVD514-3 was purified and some of it's enzymatc characteristics were investigated. The enzyme was purified about 85 fold with an overall yield of 17% from the culture medium by ammonium sulfate fractionation, Sephadex G-75 gel permeation chromatography, and CM-sephadex A-50 ion exchange chromatography. The purified enzyme functions optimally at pH 9.0 and 80℃. The enzymatic activity was increased by the presence of Mg^2+ and Mn^2 ions.

핵전이에 의한 Aspergillus oryzae var oryzae와 Penicillium verruculosum F-3의 잡종으로부터 Amylase 생산

양영기,박열,김성준,문명님,박형남,임채영,이영하 한국산업미생물학회 1994 한국미생물·생명공학회지 Vol.22 No.1

핵전이에 의해 Aspergillus oryzae var oryzae와 MPV 31 사이의 잡종을 획득하였고, 그 특성과 Amylase 활성 및 동위효소 양상을 조사한 결과는 다음과 같다. UV와 MNNG의 이중처리에 의해 A. oryzae var oryzae와 P. verruculosum F-3의 영양요구성 돌연변이를 각각 3주, 6주를 획득하였으며, 핵전이를 위한 원형질체 형성의 최적조건은 pH 5.8, 1.0% Novozym234, 0.6 M KCI 그리고 효소의 처리시간은 2시간으로 하였다. 또한 원형질체 재생을 위한 삼투안정제도 역시 0.6 M KCI이었으며, 재생율은 각각 56.4%, 52.3%로 나타났다. 핵전이에 의한 형질전환율은 3.0×10exp(-5)∼1.0×1^(-5)으로 나타났으며, 형질전환체의 유전적 안정성과 conidial size, 핵염색, 그리고 DNA 함량의 측정결과 핵형은 aneuploid로 추정되었고 total amylase의 측정 결과 형질전환체가 모균주에 비해 1.3∼2.2배 가량 증가하였으며, 동위효소 양상은 형질전환체와 모균주에서 유사하게 나타났으나 amylase 활성에 차이가 있음을 알 수 있었다. Intergeneric hybrids formed between Aspergillus oryzae var oryzae and Penicillium verruculosum F-3 were obtained by nuclear transfer technique. Several auxotrophic mutants isolated from conidio-spores of theh two strains mutagenized with ultraviolet and N-methyl-N-nitrosoguanidine. Optimal conditions for formation of intergeneric hybrids were investigated. Frequencies of hybrid formation by nuclear transfer were 3×10exp(-5)∼1×10exp(-5). From observation of genetic stability, conidial size, DNA content, nuclear stain, it was suggested that their karyptypes are aneuploid. The hybrid posses the 1.3∼2.2 fold higher amylase activities than those of parental strains. It was also revealed that some hybrids had different isozyme patterns compared to those of parental strains by amylase activity assays.

연구논문 : Aspergillus oryzae로 발효한 자색당근을 첨가한 발효유의 품질 및 관능 특성에 관한 연구

신배근 ( Bae Keun Shin ),강선아 ( Suna Kang ),한정인 ( Jung In Han ),박선민 ( Sunmin Park ) 한국식생활문화학회 2015 韓國食生活文化學會誌 Vol.30 No.3

In this study, we compared the organoleptic and other qualities of fermented milk containing 10 or 15% purple carrot extract that had either been previously fermented with Aspergillus oryzae or not fermented. Fermentation characteristics, pH, chromaticity, viscosity, viable cell counts, and sensory evaluations were measured. The pH and acid values did not differ between purple carrot extract fermented with Aspergillus oryzae and non-fermented extract. Viable cell counts were significantly higher in 15% purple carrot extract fermented with Aspergillus oryzae compared to the control after fermentation. Regarding characteristic changes, purple carrot extract fermented with Aspergillus oryzae group showed a lower red value but higher yellow value compared with non-fermented purple carrot extract due to heat-sterilization. Both fermented and non-fermented extract groups showed significantly increased viscosity compared to control. In the sensory evaluation, 15% purple carrot extract fermented with Aspergillus oryzae showed the highest score. In conclusion, addition of 15% purple carrot extract fermented with Aspergillus oryzae resulted in a superior fermented milk product.

Aspergillus oryzae로 제조한 Soybean koji 식이가 40-60대 중년여성의 손톱 개선에 미치는 영향

이성아,강상모 중소기업융합학회 2020 융합정보논문지 Vol.10 No.3

본 연구에서는 노화시작으로 손톱 상태가 나빠지는 중년 여성이 Aspergillus oryzae 로 배양한 콩 koji를 섭취하므로 손톱의 건강상태가 얼마나 개선되는지를 알아보았다. 8주 동안 40~60대 여성을 대상으로 대조군은 삶은 비 발효 콩을, 시험군은 Aspergillus oryzae로 발효한 콩 koji를 섭취하도록 하였다. 연구결과 시험군의 손톱 표면의 들뜸이나 갈라짐 상태가 개선되고, 수분량은 대조군에 비해 5.1배(p<.01), 수분 손실량은 대조군 감소량보 다 1.4(p<.05)배, 손톱두께는 대조군에 비해 1.9(p<.05)배, 표면의 주름상태는 대조군에 비해 약 2.4(p<.01)배 정도 각각 개선됨을 보였다. 따라서 콩 koji 섭취가 손톱건강상태 개선에 영향을 미치는 것으로 나타났으며. 연구를 통하여 콩 koji가 이너뷰티 소재로서 활용이 가능할 것으로 기대한다. The study was conducted to investigate the improvement nail health in middle-aged women aged 40∼60 years who intake soybean koji by incubating Aspergillus oryzae(황국균), and utilized non-fermented soybeans as a control group and soybean koji as an experiment group for eight weeks. After clinical trials, the lifting and cracking conditions of the nails in the experiment group were improved, The moisture content increased 5.1 times compared to the control group, The water loss was 1.4 times less the reduction to the control group. and nail thickness increased by 1.9 times compared to controls. Wrinkles on the surface improved by about 2.4 times compared to the control group. In conclusion, consumption of this soybean koji of A. oryzae's has been shown to have an impact on improving nail is expected to be used Therefore, I propose this soybean koji of A. oryzae 's as an inner beauty for improving nail is in middle-aged women aged 40∼60 years.

Construction of a Shuttle Vector for Heterologous Expression of a Novel Fungal a-Amylase Gene in Aspergillus oryzae

( Yanchen Yin ),( Youzhi Mao ),( Xiaolie Yin ),( Bei Gao ),( Dongzhi Wei ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.7

The filamentous fungus Aspergillus oryzae is a well-known expression host used to expresshomologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl2-mediated transformation method.As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. ThePcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30oC. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

추출조건이 chitosan의 제조에 미치는 영향

장재선 京畿專門大學 2001 京畿專門大學 論文集 Vol.- No.29

Chitosan have been usually prepared by deacetylation of chitin derived from Crustacea species such as lobster and crab with cone. NaOH. However, such treatment not only deacetylation N-acetyl glucosamine residues but also causes degradation of glycosidic bonds of chitins, decreasing their molecular weight. Some fungi contains as components of their cell walls. If chitosans prepared from these fungi have unique properties compared with those derived from Crustacea species, they would be valuable for industrial use. Chitosan was isolated from Aspergillus oryzae. This can be the potential resource for the production of chitosan. The composition of A. oryzae has been investigated. The chemical analysis of A. oryzae was consists chiefly of carbohydrate(42.8%) and protein(42.5%), with smaller amounts of moisture(6.4%), ash(4.9%) and crude fat(3.4%). In the case of isolating of chitosan from A. oryzae, hot alkali extraction(40% NaOH, 120℃, Ihr.) was carried out to remove alkali soluble glucan, protein and lipid. This was followed by acetic acid extraction(0.5 N acetic acid, 95℃, 12hr.) to extract the chitosan. To isolated chitosan from A. oryzae and the affect of the yield of chitosan-glucan complex and glucosamine, the alkaline concentration, the temperature, and the extraction time were examined by ANOVA test. At the 5% significant level, the yield of chitosan-glucan complex was only affected by the extraction time while the glucosamine content was affected by the temperature. The yield of chitosan and chitosan-glucan complex were 5.89%(dry basis) and 20.73%(dry basis) of homogenized A. oryzae, respectively.

Comparative study on metabolic changes of <i>Aspergillus oryzae</i> isolated from fermented foods according to culture conditions

Park, Min Kyung,Seo, Jeong-Ah,Kim, Young-Suk Elsevier 2019 International journal of food microbiology Vol.307 No.-

<P><B>Abstract</B></P> <P> <I>Aspergillus oryzae</I> is an important microbial starter for making diverse fermented foods due to its high hydrolytic enzyme activities. In this study, two strains of <I>A. oryzae</I> (AOB/AOK) with different activities of hydrolytic enzymes, such as α-amylase, protease and lipase, were cultured under various conditions of temperature, fermentation time, and initial pH. Comparative mass spectrometry (MS) based metabolomic analysis was performed to obtain primary metabolites and secondary volatile metabolite data sets. In the results of partial least square-discriminant analysis (PLS-DA), fatty acids and volatile metabolites derived from fatty acids and amino acids mainly contributed to AOK with higher protease and lipase activities, whereas carbohydrate-derived volatiles, sugars and sugar alcohols were related to main metabolites of AOB with higher α-amylase activity. The temperature and initial pH were critical factors for the generation of primary metabolites and secondary volatile metabolites, such as organic acids, fatty acids-derived volatiles, and some amino acids, in both <I>A. oryzae</I> strains. This study demonstrated that the specific culture conditions were closely linked to the formation of primary metabolites and secondary volatile metabolites of <I>A. oryzae</I>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Two <I>A. oryzae</I> strains were cultured under various conditions of fermentation times, temperatures, and initial pH. </LI> <LI> Primary metabolites and secondary volatile metabolites of <I>A. oryzae</I> were analyzed using GC-TOF/MS and GC–MS, respectively. </LI> <LI> The PLS-DA score plots indicated that the metabolic changes were closely linked to hydrolytic enzymatic activities. </LI> <LI> Higher temperature and lower pH can significantly affect the formation of metabolites in both <I>A. oryzae</I> strains. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

건조한 남은 음식물을 이용한 Aspergillus oryzae균주 배양조건과 그 배양물 급여가 닭의 영양소 이용률에 미치는 영향

황보 종,홍의철,이병석,배해득,김원,노환국,김재황,김인호,Hwangbo J.,Hong E. C.,Lee B. S.,Bae H. D.,Kim W.,Nho W. G.,Kim J. H.,Kim I. H. 한국가금학회 2005 韓國家禽學會誌 Vol.32 No.4

남은 음식물에서 Aspergillus oryzae(AO)의 유용한 배양조건(시간, 수분함량, 접종균수)을 구하기 위하여 2가지 실험과 닭에서 그 배양물의 영양소 이용율을 구명하기 위하여 본 시험을 수행하였다. <시험 1> 적정 배양시간과 접종균수의 측정을 위하여 수분함량을 30, 40, 50, $60\%$로 정하였다. 잔반 사료에 접종하는 seed로서 1mL Aspergillus oryzae(AO) $(1.33\times10^5\;CFU/mL)$를 이용하였다. 배양을 위한 적정 수분함량은 $40\~50\%$였으며 배양시간은 72시간 이상이었다. 계속하여 AO 균수에 의한 효과를 결정하기 위해 AO seed 0.25, 0.50, 0.75, 1.00mL를 남은음식물 사료에 접종하였다. AFW의 AO균수를 비교하기 위하여 72시간과 96시간 배양하였으나, 접종량에 따른 유의차는 없었다. 다시 수준을 낮추어 0.01, 0.05, 0.10mL를 접종하고 72시간과 96시간동안 배양한 결과, 72시간 배양 AO colony 수는 0.1 mL 접종 처리 구에서 가장 많았다. <시험 2> FW의 배양조건을 결정한 후에, 20수의 5주령 Hubbard종을 공시하여 소화율 시험을 수행하였다. 처리구는 T1-대조구, T2-기초사료 $60\%$+건조잔반 $40\%$, T3-기초사료 $60\%$+건조잔반 $20\%$+AO균 접종 건조잔반 $20\%$, T-4-기초사료 $60\%$+AO균 접종 건조잔반 $40\%$ 처리구 등 총 4처리구, 처리구당 5반복씩 20수를 인공항문 장착하여 소화율을 조사하였다. 영양소 소화율에 있어 일반성분과 아미노산은 대조구에 비해 T2 처리구가 전성분에서 낮았고(p<0.05), AFW(T3, T4)처리구에 비해 조지방과 인의 소화율에서 차이를 보였다(p<0.05). T3와 T4는 T2에 비해 조섬유 및 조회분의 소화율이 증가하였다(p<0.05). 대조구는 T3와 T4에 비해 필수아미노산인 arginine, leucine, phenylalanine의 소화율이 높았으나(p<0.05), T3와 T4 처리구는 T2 처리구에 비해 필수아미노산 arginine, lysine 및 thereonine과 비필수아미노산 alanine의 소화율이 개선되었다(p<0.05). 이러한 결과들로부터 건조 잔반의 AO 배양조건 구명과 영양소 이용율 시험을 통해 FW사료의 품질이 기초사료에 비해 영양소 이용율이 낮고, AFW 사료가 AO에 의해 개선될 수 있음을 시사한다 Two experiments were carried out to assess the appropriate incubation conditions namely; duration, moisture content and the ideal microbial inoculant for fermented dried food waste(EW) offered to broilers. The nutrient utilization of birds fed the FW diets at varying dietary inclusion rates was also compared with a control diet. In Experiment 1, different moisture contents(MC) of 30, 40, 50 and $60\%$ respectively were predetermined to establish the ideal duration of incubation and the microbial inoculant. A 1mL Aspergillus oryzae(AO) $(1.33\times10^5\;CFU/mL)$ was used as the seed inoculant in FW. This results indicated that the ideal MC for incubation was $40\~50\%$ while the normal incubation time was > 72 hours. Consequently, AO seeds at 0.25, 0.50, 0.75 and 1.00mL were inoculated in FW to determine its effect on AO count. The comparative AO count of FW incubated for 12 and 96 hours, respectively showed no significant differences among varying inoculant dosage rates. The FW inoculated with lower AO seeds at 0.10, 0.05 and 0.01mL were likewise incubated for 72 and 96 hours, respectively and no changes in AO count was detected(p<0.05). The above findings indicated that the incubation requirements for FW should be $%40\~50\%$ for 72 hours with an AO seed incoulant dosage rate of 0.10mL. Consequently, in Experiment II, after determining the appropriate processing condition for the FW, 20 five-week old male Hubbard strain were used in a digestibility experiment. The birds were divided into 4 groups with 5 pens(1 bird per pen). The dietary treatments were; Treatment 1 : Control(Basal diet), Treatment 2 : $60\%$ Basal+4$40\%$ FW, Treatment 3 : $60\%$ $Basal+20\%\;FW+20\%$ AFW(Aspergillus oryzae inoculate dried food-waste diet) and Treatment 4: $60\%$ Basal+$40\%$ Am. Digestibility of treatment 2 was lowed on common nutrients and amino acids compared with control(p<0.05) and on crude fat and phosphorus compared with AFW treatments(T3, T4)(plt;0.05). Digestibility of treatment 3 and 4 increased on crude fiber and crude ash compared treatment 2 (p<0.05). Digestibility of control was high on agrinine, leucine, and phenylalnine of essential amino acids compared with treatment 3 and 4(p<0.05), and diestibility of treatment 3 and 4 was improved on arginine, lysine, and threonine of essential amino acids. Finally, despite comparable nutrient utilization among treatments, birds fed the dietary treatment containing AO tended to superior nutrient digestion to those fed the $60\%$ Basa1+$40\%$ FW.