• Journal of Microbiology and Biotechnology
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• 제21권9호
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• pp.960-967
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• 2011

Tannin acyl hydrolase, also known as tannase, is an enzyme with important applications in the food, feed, pharmaceutical, and chemical industries. However, despite a growing interest in the catalytic properties of tannase, its practical use is very limited owing to high production costs. Several studies have already demonstrated the advantages of solid-state fermentation (SSF) for the production of fungal tannase, yet the optimal conditions for enzyme production strongly depend on the microbial strain utilized. Therefore, the aim of this study was to improve the tannase production by a locally isolated A. niger strain in an SSF system. The SSF was carried out in packed-bed bioreactors using polyurethane foam as an inert support impregnated with defined culture media. The process parameters influencing the enzyme production were identified using a Plackett-Burman design, where the substrate concentration, initial pH, and incubation temperature were determined as the most significant. These parameters were then further optimized using a Box-Behnken design. The maximum tannase production was obtained with a high tannic acid concentration (50 g/l), relatively low incubation temperature ($30^{\circ}C$), and unique low initial pH (4.0). The statistical strategy aided in increasing the enzyme activity nearly 1.97-fold, from 4,030 to 7,955 U/l. Consequently, these findings can lead to the development of a fermentation system that is able to produce large amounts of tannase in economical, compact, and scalable reactors.

• Food Science and Biotechnology
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• 제18권3호
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• pp.732-738
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• 2009

An acidic polygalacturonase (PG) from Aspergillus kawachii was produced by solid state fermentation employing a polyurethane foam support. The conditions used for the production of acidic PG were particle size of support (0.6 or 500 $mm^3$) and fermentation time. From the factors studied, the particle size had important influence on enzyme production. The best conditions for acidic PG production were $0.6\;mm^3$ particle size, 18 hr at $30^{\circ}C$ and initial pH of 5.0. In addition, pectin was extracted from citrus pomaces (grapefruit, lime, and tangerine) by acidic PG at $50^{\circ}C$ for 24 hr with citric acid solution. Infrared spectroscopy showed that lime pomace had more high-methoxylated (65%) endogenous pectin than was obtained than from grapefruit or tangerine pomaces. The enzymatically extracted pectin yield in dry basis (d.b.) for grapefruit and lime pectins were 6.95 and 4.25%, respectively. The citric acid solution alone also contributed to pectin extraction from citrus pomaces (7-9%, d.b.). Limited pectin extraction by acidic PG from tangerine pomace was most likely due to the presence of low-methoxylated endogenous pectin. The enzymatic method for pectin extraction using acidic PG from A. kawachii is a promising technique for releasing highly polymerized pectic substances from high-methoxylated lime or grapefruit pomaces.

• Restorative Dentistry and Endodontics
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• 제21권1호
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• pp.403-424
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• 1996

Cell culture methods have been used to assess the cytotoxicity of dental materials. Different paramaters are used to monitor cytotoxic effects. But it is difficult to compare each investigator's results with different methods. The objective of this study was to investigate cytotoxic effect of several retrograde filling materials according to cell lines and assay methods. Cytotoxicity of Bestalloy (Dogmyung, Korea), Prisma APH(Densply International Inc., U.S.A.), Clearfil FII (Kuraray Co., Japan), Fuji II (GC Co., Japan), Fuji II LC (GC Co., Japan) and IRM (Densply Co., U.S.A.) on L929, 3T3 and KB permanent cell lines was measured. Radiochromium, Lactate dehydrogenase (LDH) release method and colorimetric assays, namely neutral red (NR) and MTT were used. Each material was mixed according to the manufacturer's instruction. They were tested as solid and extracted state. Cell culture media were added to each mixed or solid materials then the solution was collected and used as extract solutions. Solid Fuji II showed mild cytotoxicity on three cell lines using radiochromium release method. There was no difference in cytotoxicity of extract solution group using radiochromium release method. In colorimetric assay immediate Fuji II group and all the IRM groups showed severe cytotoxic effect. Difference in cyctotoxicity was due to rather kinds of cell lines than assay methods. Solid Fuji II and IRM showed mild cytotoxicity on three cell lines. But extract solutions had different cytotoxic effect according to cell lines using LDH release assay. Light-cured glass ionomer had mild to moderate degree of cytotoxicity on three cell lines. Cytotoxicity was affected by specimen prepaton. Susceptibility of each cell ines were also affected by assay emthods. It was suggested that cytotoxicity study using only one cell line and/or assay method might not accurately reflect the real toxic nature of dental biomaterials.

• 한국산림과학회지
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• 제77권2호
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• pp.208-215
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• 1988

현사시(Populus alba ${\times}$ glandulosa) 기내배양(器內培養)한 엽육조직(葉肉組織)에서 원형질체(原形質體)를 분리(分離), 배양(培養)하여 식물체(植物體) 재분화(再分化) 과정(過程)에 관(關)해 조사(調査) 검토(檢討)하였다. $NH_4NO_3$를 뺀 MS배지(培地)에 BAP 0.5mg/l와 2.4-D 2.0mg/l를 첨가(添加)하여 liquid plating 법(法)으로 원형질체(原形質體)를 배양(培養)하였을때 비교적(比較的) 높은 세포분열(細胞分裂)이 일어났다. colony 형성율(形成率)은 gauze를 넣은 semi-solid agar plating 법(法)에서 가장 높게 일어나서 배양(培養) 5주후(週後)에는 micro-callus가 형성(形成)되었다. gauze를 제거(除去)한 후(後)에는 원형질체(原形質體) 분리배양후(分離培養後) 8주(週)가 되었을때 mini-callus가 형성(形成)되었으며 이들 callus는 2, 4-D 0.5mg/l와 BAP 0.1mg/l를 첨가(添加)한 MS배지에서 증식되었다. shoot 형성(形成)은 zeatin 1.0mg/l를 첨가(添加)한 배지에서 이식한지 3주(週)만에 일어나기 시작했으며 6주후(週後)에 뿌리발달을 도모하고져 생장조절물질(生長調節物質)이 첨가(添加)되지 않은 1/2MS배지로 이식하였다. 이식한지 4주후(週後)에 많은 뿌리가 형성(形成)되었다. 발근(發根)이 된 개체(個體)는 온실(温室)에 이식(移植)하였는데 원형질체(原形質體)를 분리(分離)하여 완전식물체(完全植物體)로 분화(分化)시켜 온실(温室)에 이식(移植)하기 까지는 22주(週)가 걸렸다.

• Journal of Plant Biotechnology
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• 제2권1호
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• pp.43-49
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• 2000

An efficient and reliable Agrobacterium transformation procedure based on TDZ (thidiazuron)-induced organogenesis was established and applied to six Brazilian eggp1ant varieties. Optimum transgenic plants recovery was achieved upon the study of the following parameters affecting transformation efficiency, using F-100 variety as a model: i) explant source; ii) pre-culture period; iii) physical state of the pre-culture medium and iv) coculture conditions. The highest frequency of kanamycin-resistant calli derived from leaf explants (5%) was obtained without a pre-culture period and co-cultivation for 24 h in liquid medium followed by five days on solid RM (regeneration medium). For cotyledon explants, best results were achieved upon a pre-culture of 24 h in liquid RM and a co-cultivation period of 24 h in liquid RM followed by three days in solid RM, resulting in a transformation Sequency of 22.7%. Kanamycin-resistant organogenic calli were also obtained from cultivars Emb, Preta Comprida, Round nose Shaded, Campineira and Florida Market. The expression pattern of an epidermis-specific promoter was studied using transformants expressing a chimaeric construct comprised by the promoter Atgrp-5 transcriptionally fused to the coding region of the gus gene. The expression pattern was similar to that previously observed in tobacco and Arabidopsis thaliana, with preferential expression at the epidermis and the stem phloem. These results support the idea that the Atgrp-5 promoter can be used to drive defense genes in these tissues, which are sites of pathogen interaction and spread, in programs for the genetic improvement of eggplant.

• 한국식품과학회지
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• 제20권5호
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• pp.666-673
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• 1988

쌀을 기질로 하는 유산균 음료 발효에서 Bacillus와 효모의 혼합배양을 이용한 고체상태 예비발효와 extruder를 이용한 압출조리 전처리가 유산균의 생육을 증진하는 효과에 대하여 검토하였다. Bacillus laevolacticus와 Saccharomyces cerevisiae의 혼합배양을 쌀과 탈지 대두분의 혼합물에 접종하여 고체 상태로 $45^{\circ}C$에서 배양한 후 자가발열형 단일축 압출성형기를 통하여 처리함으로서 살균과 조직의 변화를 도모하였다. 이렇게 처리된 물질을 분산액으로 만들어 Lactobacillus plantarum과 Leuconostoc mesenteroides 혼합배양을 접종하여 유산발효시켰다. 예비발효와 압출조리에 의하여 유산균의 증식속도와 산생성이 증가하였으며 가용성 고형분의 함량이 크게 증가하였고 분산안정성이 향상되었고 관능적 기호도도 증가하였다.

• Korean Chemical Engineering Research
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• 제52권3호
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• pp.302-306
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• 2014

목질계 분해효소 활성 증대를 위해 밀짚을 이용한 고체발효에서 주요 발효인자의 최적화를 수행하였다. Trichoderma reesei와 Aspergillus niger를 이용한 혼합배양에서 고체발효에 주요한 영향을 미친다고 알려진 배양온도, pH, 수분함량과 고체기질 크기를 순차적 최적화를 진행하였다. 실험에 적용 된 인자 모두 목질계 분해효소 활성에 유의한 효과를 주었으며, 발효온도 $40^{\circ}C$, pH 7, 수분함량 75%와 고체기질 크기 0.25~0.5 mm가 목질계 분해효소 생산을 위한 최적 조건임을 알 수 있었다. 최적조건 하에서 밀짚을 이용한 고체발효를 수행하였을 때, 효소활성 기준 cellulase 10.3 IU, endoglucanase 100.3 IU, ${\beta}$-glucosidase 22.9 IU와 xylanase 2261.7 IU/g dry material을 배양 96시간에 확인할 수 있었다. 본 결과는 기존 효소활성 대비 각각 72.6, 48.7, 55.2와 51.9% 증가한 수치로 혼합배양과 순차적 최적화를 적용하여 효과적인 목질계 분해효소 활성 증대가 가능함을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제23권7호
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• pp.1004-1014
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• 2013

Six nonpathogenic fungal strains isolated from alkaline soils of Buenos Aires Province, Argentina (Acremonium murorum, Aspergillus sidowii, Cladosporium cladosporoides, Neurospora tetrasperma, Purpureocillium lilacinum (formerly Paecilomyces lilacinus), and Westerdikella dispersa) were tested for their ability to produce keratinolytic enzymes. Strains were grown on feather meal agar as well as in solid-state and submerged cultures, using a basal mineral medium and "hair waste" as sole sources of carbon and nitrogen. All the tested fungi grew on feather meal agar, but only three of them were capable of hydrolyzing keratin, producing clear zones. Among these strains, P. lilacinum produced the highest proteolytic and keratinolytic activities, both in solid-state and submerged fermentations. The medium composition and culture conditions for the keratinases production by P. lilacinum were optimized. Addition of glucose (5 g/l) and yeast extract (2.23 g/l) to the basal hair medium increased keratinases production. The optimum temperature and initial pH for the enzyme production were $28^{\circ}C$ and 6.0, respectively. A beneficial effect was observed when the original concentration of four metal ions, present in the basal mineral medium, was reduced up to 1:10. The maximum yield of the enzyme was 15.96 $U_c/ml$ in the optimal hair medium; this value was about 6.5-fold higher than the yield in the basal hair medium. These results suggest that keratinases from P. lilacinum can be useful for biotechnological purposes such as biodegradation (or bioconversion) of hair waste, leading to a reduction of the environmental pollution caused by leather technology with the concomitant production of proteolytic enzymes and protein hydrolyzates.

• Applied Biological Chemistry
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• 제15권3호
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• pp.199-206
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• 1972

Aspergillus awamori가 생산하는 konjak mannan 분해 효소군(酵素郡)에 관한 연구로서 효소생산조건, 효소의 정제 및 성질, 효소분해에 미치는 감마선 조사(照射)의 영향을 조사하여 다음과 같은 결과를 얻었다. 1) 보존균주(保存菌株) 80여개중 konjak mannan 분해효소를 다량 생산하는 것으로 A. awamori를 선정하였다. 2) A. awamori를 밀기울 고체배양하는 경우 최적 배양일수는 3일, 살수(撒水)의 pH는 4, 살수량(撒水量)은 100%가 가장 좋았다. 3) A. awamori를 액체 진탕 배양하는 경우 최적 배양일수는 6일, 기본배지(基本培地)에 대한 추가탄소원(追加炭素源)으로는 xylose 0.1%, 질소원(窒素源)으로는 peptone 0.04%가 가장 좋았다. 4) A. awamori의 고체배양물에서 konjak mannan분해효소를 유안염석(硫安鹽析) 및 DEAE-Sephadex column chromatography에 의하여 정제(精製)하고 fraction I과 fraction II를 분리하였다. 5) fraction I과 fraction II의 작용최적 pH는 모두 4이었고 pH 안정(安定)범위는 각각 $3.5{\sim}5$ 및 $3{\sim}6$이었으며 konjak mannan에 대한 가수분해도는 각각 9%, 50%, guar gum에 대한 것은 각각 14%, 11%이 었 다. 6) 조효소액(粗酵素液)에 의한 konjak mannan의 가수분해도는 0.5 Mrad이상의 감마선 조사(照射)에 의하여 약간 촉진되었으며 특히 수화(水和)상태에서의 조사(照射)에 의하여 그 효과가 현저하였다. 7) konfak mannan의 감마선 조사(照射)는 점도(粘度)강하와 환원력(還元力)증가를 초래하였으며 건조상태보다는 수화(水和) 상태에서 그 효과가 현저하였다.

• KSBB Journal
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• 제23권4호
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• pp.345-349
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• 2008

본 연구는 에탄올 생산 부산물인 주정박의 사료로서의 가치를 향상시키고 효소 활성을 유지하면서 아미노산이 다량 함유된 발효 사료를 개발하기 위한 고체 발효 조건을 최적화하는데 목적을 두었다. 사용된 균주의 pH에 대한 영향을 살펴본 결과, pH 4에서 효소 활성이 우수하였으며 또한 이 조건은 낮은 pH 조건이므로 잡균에 대한 오염도 예방 할 수 있어 본 실험의 최적 액체배양 조건임을 확인할 수 있었다. 고체 배양을 위한 배양 조건 탐색에서는 60%의 수분을 함유한 고체 배양에서 가장 좋은 효소 활성의 결과를 나타내었으며 적정 배지 조성을 위한 혼합 비율 탐색의 경우 밀기울 함량이 높고 DDG 함량이 낮을수록 효소 활성은 좋았으나 아미노산 함량은 낮은 반면, DDG 함량이 높고 밀기울 함량이 낮을수록 효소 활성은 낮았지만 아미노산 함량은 높은 결과를 나타내었다. 따라서 효소활성 ($\geqq$ 1,000 U/g) 및 아미노산 함량 ($\geqq$ 28%)이 적당한 고체 발효 배지 조성의 비율은 DDG와 밀기울이 1 : 4였다. 이렇게 해서 얻어진 결과로 약 1 ton 정도의 발효 사료 시제품을 생산하였으며 시제품의 효소활성과 조단백질 함량은 각각 1,024 U/g과 33.6%였다.