• KSBB Journal
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• 제27권6호
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• pp.352-360
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• 2012

Bacillus clausii I-52 which produced SDS- and $H_2O_2$-tolerant extracellular alkaline protease (BCAP) was isolated from heavily polluted tidal mud flat of West Sea in Incheon, Korea and stable strain (transformant C5) of B. clausii I-52 harboring another copy of BCAP gene in the chromosome was developed using the chromosome integration vector, pHPS9-fuBCAP. When investigated the production of BCAP using B. clausii transformant C5 through pilot-scale submerged fermentation (500 L) at $37^{\circ}C$ for 30 h with an aeration rate of 1 vvm and agitation rate of 250 rpm, protease yield of approximately 105,700 U/mL was achieved using an optimized medium (soybean meal 2%, wheat flour 1%, sodium citrate 0.5%, $K_2HPO_4$ 0.4%, $Na_2HPO_4$ 0.1%, NaCl 0.4%, $MgSO_4{\cdot}7H_2O$ 0.01%, $FeSO_4{\cdot}7H_2O$ 0.05%, liquid maltose 2.5%, $Na_2CO_3$ 0.6%). The enzyme stability of BCAP was increased by addition of polyols (10%, v/v) and also, the stabilities of BCAP towards not only the thermal-induced inactivation at $50^{\circ}C$ but also the SDS and $H_2O_2$-induced inactivation at $50^{\circ}C$ were enhanced. Among the polyols examined, the best result was obtained with propylene glycol (10%, v/v). The BCAP supplemented with propylene glycol exhibited extreme stability against not only the detergent components such as ${\alpha}$-orephin sulfonate (AOS) and zeolite but also the commercial detergent preparations. The granulized enzyme of BCAP was prepared with approximately 1,310,000 U/g of granule. Wash performance analysis using EMPA test fabrics revealed that BCAP granule exhibited high efficiency for removal of protein stains in the presence of anionic surfactants as well as bleaching agents. When compared to Savinase 6T$^{(R)}$ and Everlase 6T$^{(R)}$ manufactured by Novozymes, BCAP under this study probably showed similar or higher efficiency for the removal of protein stains. These results suggest that the alkaline protease produced from B. clausii transformant C5 showing high stability against detergents and high wash performance has significant potential and a promising candidate for use as a detergent additive.

• Journal of Microbiology and Biotechnology
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• 제21권6호
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• pp.627-636
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• 2011

A commercially important alkaline protease, produced by Bacillus sp. RRM1 isolated from the red seaweed Kappaphycus alvarezii (Doty) Doty ex Silva, was first recognized and characterized in the present study. Identification of the isolated bacterium was done using both biochemical characterization as well as 16S rRNA gene sequencing. The bacterial strain, Bacillus sp. RRM1, produced a high level of protease using easily available, inexpensive agricultural residues solid-state fermentation (SSF). Among them, wheat bran was found to be the best substrate. Influences of process parameters such as moistening agents, moisture level, temperature, inoculum concentration, and co-carbon and co-nitrogen sources on the fermentation were also evaluated. Under optimized conditions, maximum protease production (i.e., 2081 U/g) was obtained from wheat bran, which is about 2-fold greater than the initial conditions. The protease enzyme was stable over a temperature range of 30-$60^{\circ}C$ and pH 6-12, with maximum activity at $50^{\circ}C$ and pH 9.0. Whereas the metal ions $Na^+$, $Ca^{2+}$, and $K^+$ enhanced the activity of the enzyme, others such as $Hg^{2+}$, $Cu^{2+}$, $Fe^{2+}$, $Co^{2+}$, and $Zn^{2+}$ had rendered negative effects. The activity of the enzyme was inhibited by EDTA and enhanced by $Cu^{2+}$ ions, thus indicating the nature of the enzyme as a metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents, surfactants, and organic solvents. Moreover, the present findings opened new vistas in the utilization of wheat bran, a cheap, abundantly available, and effective waste as a substrate for SSF.

• 한국의류학회지
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• 제24권1호
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• pp.96-104
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• 2000

The purpose of this study was to investigate the antimicrobial activity, antimutagenic and anticancer effects and dyeing properties of the fermented indigo extract. The physiological effects of natural color extracts from colorant plants(gardenia, beet and indigo) were studied. The methanol extract of indigo showed an inhibitory effect on the growth of E. coli and Staph. aureus, and also showed a strong antimicrobial effect on Trich. mentagrophytes compared to others. The methanol extract of indigo showed antimutagenic activities against aflatoxin B1(AFB1) in the Ames test using Salmonella typhimurium TA 100. The proliferation of Clone M-3 mouse melanoma cells and A431 human epidermoid carcinoma cells was inhibited by the methanol extract of indigo. So we decided to use natural indigo for dyeing the fabrics because of those effects. Dried indigo leaves were fermented at variouss temperature and the fermented indigo was reduced by using alkaline(NaOH, Ca(OH)2) and glucose to dye the fabrics. The values of K/S fermented indigo showed the highest value when it was fermented at 3$0^{\circ}C$. The indigo fermented at 3$0^{\circ}C$ had the greatest number of total bacterial counts and we identified one of the main microorganisms as Aspergillus niger. This microorganism was responsible for the indigo fermentation and accelerated indigo fermentation. So it can be supposed to reduce the fermentation period of indigo by inoculating Aspergillus niger into the indigo leaves at 3$0^{\circ}C$.

• Asian-Australasian Journal of Animal Sciences
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• 제15권5호
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• pp.665-670
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• 2002

The expediency of replacing cost prohibitive and often inaccessible traditional protein supplements prompted the monitoring of hematological parameters was carried out in female goats at 0, 30, 60 and 90 days post feeding. Rumen environment was (3), respectively fed supplements containing either a leaf meal mixture (LMTM) of Leucaena leucocephala-Morus alba-Tectona grandis (2:1:1) or traditional protein supplements groundnut cake (GNC) or soybean meal (SBM) and wheat straw as basal diet. The periodic monitoring of hematological parameters was carried out in female goats at 0, 30, 60 and 90 days post feeding. Rumen environment was studied in bucks in a $3{\times}3$ switch over design. Rumen liquor was collected at 0, 2, 4, 6 and 8 h post feeding after 4 weeks of feeding. The goats fed on LMTM or GNC had similar dry matter intake (g/kg $W^{0.75}$), which was significantly (p<0.05) higher than SBM. Except for packed cell volume (PCV), none of the blood biochemical constituents (Hemoglobin, serum glucose, total protein, serum albumin (A) and globulin(G), A:G ratio, alkaline phosphatase, transaminases) varied significantly due to replacement of 50% dietary protein by LMTM throughout the experiment. GNC group had significantly higher level of PCV than other treatments. However, the level of serum total protein (p<0.01) tended to increase from 60th day onwards irrespective of dietary treatments. The average rumen pH was significantly higher (p<0.001) on SBM followed by LMTM and GNC, respectively. Total volatile fatty acid (TVFA) production was comparable in goats given LMTM or GNC supplements, the corresponding values were significantly different (p<0.001) when compared with SBM. The ammonical-N, total-N and TCA-precipitable-N (mg/100 ml SRL) did not differ significantly among dietary treatments. It may be concluded that supplementing wheat straw with LMTM based concentrate had no adverse effect on voluntary intake, blood biochemical profile and rumen fermentation pattern of the goats.

• Asian-Australasian Journal of Animal Sciences
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• 제32권1호
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• pp.72-81
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• 2019

Objective: To determine the effect of gut pH and rumen microbial fermentation on glycerol encapsulated in alginate and alginate-chitosan polymers. Methods: Glycerol was encapsulated at 2.5%, 5%, 7.5%, or 10% (w/w) with sodium alginate (A) and alginate-chitosan (AC) polymers. Surface morphology and chemical modifications of the beads were evaluated using scanning electron microscopy and Fourier transform infrared (FTIR) spectra. Encapsulation efficiency was determined at the 5% glycerol inclusion level in two experiments. In experiment 1, 0.5 g of alginate-glycerol (AG) and alginate-chitosan glycerol (ACG) beads were incubated for 2 h at $39^{\circ}C$ in pH 2 buffer followed by 24 h in pH 8 buffer to simulate gastric and intestinal conditions, respectively. In experiment 2, 0.5 g of AG and ACG beads were incubated in pH 6 buffer at $39^{\circ}C$ for 8 h to simulate rumen conditions. All incubations were replicated four times. Free glycerol content was determined using a spectrophotometer and used to assess loading capacity and encapsulation efficiency. An in vitro experiment with mixed cultures of rumen microbes was conducted to determine effect of encapsulation on microbial fermentation. Data were analyzed according to a complete block design using the MIXED procedure of SAS (SAS Institute, Cary, NC, USA). Results: For AG and ACG, loading capacity and efficiency were 64.7%, 74.7%, 70.3%, and 78.1%, respectively. Based on the FTIR spectra and scanning electron microscopy, ACG treatment demonstrated more intense and stronger ionic bonds. At pH 6, 36.1% and 29.7% of glycerol was released from AG and ACG, respectively. At pH 2 minimal glycerol was released but pH 8 resulted in 95.7% and 93.9% of glycerol released from AG and ACG, respectively. In vitro microbial data show reduced (p<0.05) fermentation of encapsulated glycerol after 24 h of incubation. Conclusion: The AC polymer provided greater protection in acidic pH with a gradual release of intact glycerol when exposed to an alkaline pH.

• 한국미생물·생명공학회지
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• 제49권1호
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• pp.45-56
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• 2021

Improved amylases were developed from protoplast fusants of two amylase-producing Aspergillus species. Twenty regenerated fusants were screened for amylase production using Remazol Brilliant Blue agar. Crude enzyme extracts produced by solid state fermentation of rice bran were assayed for activity. Three variable factors (temperature, pH and enzyme type) were optimized to increase the amylase activity of the parents and selected fusants using rice bran medium and solid state fermentation. Analysis of this optimization was completed using the Central Composite Design (CCD) of the Response Surface Methodology (RSM). Amylase activity assays conducted at room temperature and 80℃ demonstrated that Aspergillus designates, T5 (920.21 U/ml, 966.67 U/ml), T13 (430 U/ml, 1011.11 U/ml) and T14 (500.63 U/ml, 1012.00 U/ml) all exhibited improved function making them the preferred fusants. Amylases produced from these fusants were observed to be active over the entire pH range evaluated in this study. Fusants T5 and T14 demonstrated optimal activity under acidic and alkaline conditions, respectively. Fusants T13 and T14 produced the most amylase at 72 h while parents TA, TC and fusant T5 produced the most amylase after 96 h of incubation. Response surface methodology examinations revealed that the enzyme from fusant T5 was the optimal enzyme demonstrating the highest activity (1055.17 U/ml) at pH 4 and a temperature of 40℃. This enzyme lost activity with further increases in temperature. Starch hydrolysis using fusant T5 gave the highest yield of glucose (1.6158 g/100 ml). The significant activities of the selected fusants at 28 ± 2℃ and 80℃ and the higher sugar yields from cassava starch hydrolysis over their parental strains indicate that it is possible to improve amylase activity using the protoplast fusion technique.

• 한국식품영양과학회지
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• 제38권1호
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• pp.76-82
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• 2009

본 연구에서는 발효과정 중 누룩의 일반성분 및 효소 활성변화를 측정하고, 단백질의 분해정도와 밀 단백질에 민감한 환자의 혈청과 반응성을 관찰하였다. 누룩의 수분함량은 발효 1일 24.20%였으며 발효가 진행됨에 따라 미생물의 이용과 증발로 인해 감소되어 발효 15일에는 13.60%를 나타내었다. 누룩 발효 중 총당과 환원당의 함량은 발효시간이 경과함에 따라 증가하는 경향을 나타내었다. 누룩의 발효 초기 조단백질 함량은 1.58%였으나 발효가 진행됨에 따라 다소증가하여 발효 15일에는 2.05%로 나타났다. 또한 누룩의 조지방 함량은 발효 초기 1.32%였으며 발효 15일에는 2.24%로 다소 증가함을 나타내었다. ${\alpha}$-Amylase 활성은 발효 초기 1,416.67 U/g에서 발효 15일에는 2,833.00 U/g으로 급격히 증가하였으며, glucoamylase 활성은 발효 초기 497.90 U/g에서 발효 10일 경과 후 705.40 U/g으로 최대 활성을 보였으나 그 후 완만한 감소를 나타내었다. 누룩의 산성 protease 활성은 발효 초기에는 검출되지 않았으나 발효 10일에 75.00 U/g으로 급격히 증가하기 시작하여 발효 15일에는 327.00 U/g에 도달하였으며 중성 protease 활성도 발효 10일에 90.00 U/g으로 급격히 증가하기 시작하여 15일에는 354.00 U/g로 검출되었으며 이후에도 계속 증가하였다. 반면에 알칼리성 protease 활성은 검출되지 않았다. 발효가 진행됨에 따라 누룩 중 고분자 단백질이 각종 미생물에 의해 분해되어 저분자의 펩타이드 밴드가 생성되었으며 밀 민감성 환자의 혈청과 반응하는 밴드도 점차 감소하여 발효 15일에는 거의 검출되지 않았으므로 누룩 중 밀 단백질의 항원성이 감소하였음을 확인할 수 있었다. 따라서 본 연구를 통해 누룩은 영양학적 측면뿐만 아니라 효소학적 측면의 장점까지 겸비한 효율적인 한국 고유의 발효 식품용 스타터(starter)로써 전통주 제조에 필수적이며, 누룩의 고분자 단백질이 발효에 의해 저분자 펩타이드로 분해됨을 확인함으로써 밀 민감성 환자들을 위한 다양한 가공식품용 소재로 활용이 기대된다.

• 한국미생물·생명공학회지
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• 제28권6호
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• pp.322-328
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• 2000

To screen agent for the treat-ment of Alzhimers Disease several strains of bacteria producing acetylcholinesterase inhibitor ware isolated from soil. Strain 960903 showed strong acetylcholinesteras inhibitory activity and low butyrylcholinesterse inhibitory activity. The strain 960903 was identified as Pseudomonas sp. Acetylcholinesterase inhibitor ws highly achieved in fermentation medium containing soluble starch 3.0%, glycerol 1.0%, pharmamedia 0.5%, KCI 0.3%, $CaCO_3$ 0.2%, MgS $O_4$..$7H_2$O 0.05%, $KH_2$$PO_4$ 0.05%(pH6.5) at $30^{\circ}C$ for 4 days. Acetylcholinesterase inhibitor was purified by Diaion WA-30($OH^{-}$) column charomatography and cellulose column chromatography. Acetylcholinesterase inhibi-tor showd the maximum wavelength at 205 nm and was soluble in water, acetic acid, ethanol, methanol and dime-thyl sulfoxide. The concentration of 50% inhibition($IC_{50}$) of inhibitor against acetylcholinesterase was 25$\mu\textrm{g}$/ml. The inhibitor was inactivated on heating ar $100^{\circ}C$ fro 15 min and more stable in acidic region than alkaline region.n.

• 한국수소및신에너지학회논문집
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• 제26권1호
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• pp.71-78
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• 2015

This study was performed to optimize the integrated thermal-alkali pre-treatment of flotation scum for the enhanced biodegradability. The optimum conditions of the integrated thermal-alkali pre-treatment were obtained using response surface methodology. The disintegration degree of carbohydrate (69.2%) and protein (57.3) were estimated under the optimum conditions. Although the optimum conditions were different, the disintegration degrees were similarly. A fermentative hydrogen batch test was conducted to evaluate the hydrogen production from scum with and/or without. The maximum hydrogen production from scum with pre-treatment was of 0.64 mol H2/mol hexoseadded, which about 1.4 times higher than without pre-treatment.

• 한국미생물·생명공학회지
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• 제5권1호
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• pp.18-23
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• 1977

섬유소자화세균 Cellulomonas flavigena KIST 321로 섬유소 단세포 단백을 생산하기 위하여 우리주변에서 제기되고 있는 각종 섬유소폐기물을 수집하여 기질로서의 사용 가능성을 검토하였는 바 그 결과를 요약하면 다음과 같다. 1. 당유를 유일한 탄소원으로 하였을 때 cellulose와 rylose에서 균체량이 가장 많았다. 2. 폐지유와 폐의유를 기질로 하였을 때 알카리 전처리를 함으로써 알카리 전처리하지 않았을 때보다 균체량이 많았으며, 폐신문지와 같은 인쇄물에서는 균체량이 적었다. 3. 짚류(straws)를 기질로 하였을 때는 대체로 균체수율이 높았으며, 볏짚, 채종대, 피를 기질로 하였을 때 균체량이 많았다. 4. 목재유의 톱밥에서는 균체량이 적었으나 잎류(leaves)에서는 비교적 많았다. 5. 기질의 종류에 따라 전처리시 NaOH농도를 다르게 하여 균체생산량을 조사하였다. NaOH 농도는 기질의 종류에 따라 최적농도가 달랐으나 짚류(Straws)에서는 0.8∼1.0％ 일때가 좋았고, 잎류(leaves)에서는 0.4∼0.6％일때가 좋았다. 6. 각종기질을 알카리 처리후 세척하여 기질로 사용하였을 때 볏짚에서는 균체량이 감소하였으나 소나무 톱밥, 피나무 톱밥, 소나무잎, 폐신문지에서는 증가하였다.