Earticle

In this study, we investigated the effect of solid culture medium on the production of cordycepin in Cordyceps militaris. The regression equation was expressed as follows: Y1= 755.3-58.6625X1+4.79432E-14X2-46.6625X3-5.66269E-14X1X2- 0.025X1X3+1.62475E-14X2X3-160.6625X1 2+0.0125X2 2-206.9625X3 2, where, Y represents the value of cordycepin content (μg/g), X1 corresponds to the weight of M. alternatus in solid culture medium (g/bottle), X2 to the water content of the solid culture medium (%), and X3 to the culture period (day). The solid culture medium was optimized using the response surface methodology, and the optimal medium composition was as follows: the weight of M. alternatus in solid culture medium and water content were 16.2% and 100.7% (20.14 mL water/20 g solid culture medium), respectively, with a culture period of 39 days. Under these conditions, the cordycepin content of the fruiting bodies reached 150.0 μg/g (actual value). The supplementation of M. alternatus in solid culture for improved cordycepin content of C. militaris seems to be a promising alternative to wild and solid cultivation.

Monacolin K (MK) was produced on soybean using Monascus ruber GM011 by a two-stage-fermentation process. The optimal temperature was identified as 28℃. Higher yield was obtained by multiple-level-temperature cultivation than by single-level-temperature cultivation. The highest yield of total MK, 4.810 mg/g dry soybean product, was attained after 30 days of solid-state fermentation. No citrinin could be detected in the fermented soybean.

Mucilage containing r-polyglutamic acid (PGA) was efficiently generated by the solid-state fermentation (SSF) of soybean grit by Bacillus firmus NA-1. B. firmus NA-1 was shown to be a glutamate-dependent strain for PGA production. The SSF of soybean grit was optimized in order to produce mucilage with a fortification of 5% glutamate, resulting in higher levels of mucilage production (6.14%) and a higher consistency index (1.1 Pa secn). The sticky mucilage was comprised of 38% PGA, 7% levan, and some biopolymers. With regard to the viscoelastic properties of the mucilage solution, the viscous modulus (G") obtained from soybean grit fortified with 5% glutamate was approximately 64 times higher titan that of the mucilage solution obtained without glutamate. Although the addition of glutamate in the SSF of soybean grit influenced the rate of PGA production, the molecular weight of PGA remained unaltered, and was detected in a range between 1,400-1,440 kDa.

Soybean grits, fortified with various bioactive components, were produced by solid-state fermentation using Bacillussubtilis HA. α-Amylase activity gradually increased during fermentation over 5days. Fibrinolytic and protease activities werehighest in the soybean grits fermented for 7days. The grits fermented for 5 days also showed the highest tyrosine content,indicating a higher peptide content. Peptides of low molecular weight (below 3,000 daltons) and browning pigments increasedwith increasing fermentation time. The fermented soybean grits showed higher contents of total phenolic compounds, toapproximately 18mg/g. DPPH free radical scavenging effects were higher in the soybean grits fermented for 3days. Also,ABTS radical scavenging effects were greater in the fermented grits compared to the unfermented grits. Overall, the soybeangrits fermented by solid-state fermentation for 5days showed enhanced production of bioactive compounds and greaterantioxidant properties.

Palm kernel cake(PKC), a by-product of palm oil industry, is a potential feed source for poultry. However, its use as poultry feed is limited due to its high fiber(mannan) content. Therefore, we have developed a bioconversion process to improve the nutritive value of PKC as poultry feed. Previously, we showed successful bioconversion of PKC by solid-state fermentation with novel manannase producing strains. In this study, the effect of gamma-irradiation of PKC on solid-state fermentation by Bacillus subtilis MBS1 has been investigated. The irradiation itself induced release of reducing sugar and soluble protein from PKC, showing structural changes or decomposition of the substrate by gamma-ray. Neutral detergent fiber content of the irradiated PKC was decreased with an increase in the irradiation dose. The level of mannanase produced during fermentation of irradiated PKC was much higher than that of untreated substrate. Compared with untreated PKC, the irradiated substrate showed higher decrease in hemicellulose and cellulose fractions after fermentation by Bacillus subtilis MBS1. This indicated the irradiation treatment of PKC enhanced degradation of mannan and other anti-nutritive polysaccharides by Bacillus subtilis MBS1. These results suggest that gamma-irradiation can be effectively used to enhance bioconversion of PKC for improving its nutritive value as poultry feed.

In this study, we optimized the production of r-polylutamic acid (PGA) in soybean milk cakes (SMC) fermented with Bacillus subtilis GT-D and B. subtilis KU-A, to be utilized as a functional food ingredient. PGA production was dependent upon the glutamate content, fermentation time, and type of Bacillus sp. The consistencies of the SMCs fermented by B. subtilis GT-D and B. subtilis KU-A were highest after 36 hr of fermentation, and then decreased gradually. The SMC fermented by B. subtilis KU-A had a higher consistency than the SMC fermented by B. subtilis GT-D. In the presence of 10% defatted soy flour (DFS), 5% glutamate in the SMC was efficiently converted into polyglutamic acid (PGA) for 24 hr, indicating a conversion yield above 96%, but its conversion then decreased with higher concentrations of glutamate. The soluble solid content (mucilage) of the SMC fermented with B. subtilis KU-A was 9.5%(w/w), and composed of 65.6% PGA (Mw 1,536 kDa) and some polysaccharides. However, the SMC fermented with B. subtilis GT-D had a mucilage content of 7.8%(w/w), and was composed of 66.4% PGA (Mw 1,409 kDa), 11.5% levan, and some polysaccharides. The viscoelastic values of the mucilage obtained using B. subtilis KU-A were much higher than those of mucilage obtained using B. subtilis GT-D. Also, the G'-value (elastic modulus) was higher than the G"-value (viscous modulus).

Mevinolin, a fungal metabolite, is a potent inhibitor of 3-hydroxy-methyl-3-glutaryl-coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme in cholesterol biosynthesis. In this investigation, the optimum factors for mevinolin production by Monascus pilosus IFO 480 in soymeal fermentation were studied. The highest yield of mevinolin, 2.82 mg mevinolin per g dry weight, without citrinin (a toxic fungal secondary metabolite) was obtained after 21 days of fermentation at 30℃ at 65% moisture content, particle size 0.6-0.9 mm, and initial substrate pH of 6.0. Mevinolin was present in the fermentation substrate predominantly in the hydroxycarboxylate form (open lactone, 92.1-97.3%), which is currently being used as a hypocholesterolemic agent.

An acidic polygalacturonase (PG) from Aspergillus kawachii was produced by solid state fermentation employinga polyurethane foam support. The conditions used for the production of acidic PG were particle size of support (0.6 or 500mm3) and fermentation time. From the factors studied, the particle size had important influence on enzyme production. Thebest conditions for acidic PG production were 0.6mm3 particle size, 18 hr at 30oC and initial pH of 5.0. In addition, pectinwas extracted from citrus pomaces (grapefruit, lime, and tangerine) by acidic PG at 50oC for 24 hr with citric acid solution.Infrared spectroscopy showed that lime pomace had more high-methoxylated (65%) endogenous pectin than was obtainedthan from grapefruit or tangerine pomaces. The enzymatically extracted pectin yield in dry basis (d.b.) for grapefruit and limepectins were 6.95 and 4.25%, respectively. The citric acid solution alone also contributed to pectin extraction from citruspomaces (7-9%, d.b.). Limited pectin extraction by acidic PG from tangerine pomace was most likely due to the presence oflow-methoxylated endogenous pectin. The enzymatic method for pectin extraction using acidic PG from A. kawachii is apromising technique for releasing highly polymerized pectic substances from high-methoxylated lime or grapefruit pomaces.

본 연구는 에탄올 생산 부산물인 주정박의 사료로서의 가치를 향상시키고 효소 활성을 유지하면서 아미노산이 다량 함유된 발효 사료를 개발하기 위한 고체 발효 조건을 최적화 하는 데 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 있어본 실험의 최적 액체배양 조건임을 확인할 수 있었다. 고체 배양을 위한 배양 조건 탐색에서는 60%의 수분을 함유한 고체배양에서 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 (≧ 1,000 U/g) 및 아미노산 함량(≧ 28%)이 적당한 고체 발효 배지 조성의 비율은 DDG와 밀기울이 1：4 였다. 이렇게 해서 얻어진 결과로 약 1 ton 정도의 발효 사료 시제품을 생산하였으며 시제품의 효소활성과 조단백질 함량은 각각 1,024 U/g과 33.6%였다.

To enhance the value as a feedstuff of distiller's dried grain (DDG) and develop fermented feedstuff, we investigated the effects of the culture conditions affecting glucoamylase activity, such as pH in submerged culture and moisture content in solid-state culture. Also, we investigated the optimal mixing ratio of DDG and wheat bran for the production of fermented feedstuff containing high content of amino acids. In culture conditions for high fermented activity, pH and moisture were optimum at pH 4 and 60%, respectively. In the case of mixing ratio, the glucoamylase activity was decreased with increase of DDG content. On the other hand, the content of crude protein was increased slowly. For the development of fermented feedstuff, the optimal mixing ratio of DDG and wheat bran was 1 to 4. Finally, we could produce approximately 1 ton (dry matter) of trial product in incubator of pilot-scale. The glucoamylase activity and the crude protein content were 1,024 U/g and 33.6%, respectively.

The growth characteristics and production of color pigments by Monascus strains were investigated during liquid culture, and production of monacolin K in red mold rice was carried out by solid state fermentation. Four different Monascus ruber strains were cultured in potato dextrose yeast extract broth (PDYB) medea at 25oC for 15 days. The high producing strain for red pigment was not corresponded to the strain for yellow pigment. Production of red pigment was high in the strain causing the fast pH change in culture broth. Production of monacolin K in red mold rice by solid state fermentation was influenced by a combination of wet cell weight and spore density in inoculum by liquid culture. Most strains showed the high production of monacolin K in red mold rice, when submerged fermentation was carried out for 5 days as inoculum for solid state fermentation. These results suggest that submerged fermentation period of inoculum have an effect on the production of monacolin K in red mold rice by solid state fermentation, and monacolin K in red mold rice could be increased by controlling the condition of submerged fermentation for inoculum.

Leaves of Aloe vera Linne and bloods of domestic animal were composted in a soild state fermentation reactor (SSFR) by using microbial additive including a bulking and moisture controlling agent. From solid-culture of microbial additive, 10 species of bacteria and 10 species of fungi were isolated and, their enzyme activities including amylase, carboxy methyl cellulase CMCase, lipase and protease were detected. Optimum fermentation conditions of Aloe leaves and domestic animal bloods in SSFR were obtained from the studies of response surface analysis employing microbial additive content, initial moisture content, and fermentation temperature as the independent variables. The optimum conditions for SSFR using Aloe leaves were obtained at 9.45$\pm$73%(w/w) of microbial additives, 62.73$\pm$4.54%(w/w) of initial moisture content and 55.32$\pm$3.14$^{\circ}C$ of fermentation temperature while those for SSFR using domestic animal bloods were obtained at 10.25$\pm$2.04%, 58.68$\pm$4.97% and 57.85$\pm$5.$65^{\circ}C$, respectively. Composting process in SSFR was initially proceeded through fermentation and solid materials were decomposed within 24 hours by maintaining higher moisture level, and maturing and drying steps are followed later. After the fermentation step, the concentrations of solid phase inorganic components were increased while that of organic components were decreased. Also, concentrations of total organic carbon(TOC), peptides, amino acids, polysaccharides, and low fatty acids in water extracts were increased. As fermentation in composting process depends on initial C/N ratios in water extracts of two samples were increased because of increased water-soluble TOC. From these results, it was revealed that solid state fermentation reactor using microbial additives can be used in composting process of organic wastes with broad C/N ratio.

The antibiotic meroparamycin was produced in the free culture system of Streptomyces sp. strain MAR01. Five solid substrates (rice, wheat bran, Quaker, bread, and ground corn) were screened for their ability to support meroparamycin production in solid-state fermentation. In batch culture, wheat bran recorded the highest antibacterial activity with the lowest residual substrate values. The highest residual substrate values were recorded for both ground corn and Quaker. On the other hand, no antibacterial activity was detected for rice as a solid substrate. The use of the original strength of starch-nitrate medium in the solid-state fermentation gave a lower antibacterial activity compared with the free culture system. Doubling the strength of this medium resulted in the increase in the activity to be equivalent to the free culture. The initial pH (7.0) of the culture medium and 2 ml of spore suspension (1 ml contains $5{\times}10^{9}spores/ml$) were the optima for antibiotic production. The water was the best eluent for the extraction of the antibiotic from the solid-state culture. Ten min was enough time to extract the antibiotic using a mixer, whereas, 60 min was required when shaking was applied. Semicontinuous production of meroparamycin using a percolation method demonstrated a more or less constant antibacterial activity over 4 runs ($450-480{\mu}g/ml$). The semicontinuous production of the antibiotic was monitored in a fixed-bed bioreactor and the maximum activity was attained after the fourth run ($510{\mu}g/ml$) and the overall process continued for 85 days.

The concept of Solid-State Fermentation is briefly explained in comparison to other fermentation principles, and several types of fermenters are presented. A recently developed "Swing Reactor" for SSF is shown. When inoculated on rice, the mould Monascus purpureus forms red pigments, Which can be used as food colors (Ang-kak, Red Rice). By Response Surface Methodology, serveral factors have been optimized for maximal red colour formation. Showing that presoaking time of rice, pH of soaking water, age of preculture and inoculum size were not of importance within the observed limits. For a fermentation time of 7 days, start humidity is optimal at 34% and temperature is optimal at 28.8 C. These results of small scale fermentation could be transferred to the Swing Reactor.

The production of conidia of entomopathogenic Beauveria bassiana by solid-state fermentation was studied for the development of a biocontrol agent against aphid Myzus persicae. The optimal conditions for conidia production on polished white rice were 40% moisture content, $25^{\circ}C$ culture temperature, 2-day-old seeding culture grown in 3% corn meal, 2% rice bran, 2% corn steep powder medium, initial conidia concentration of $10^7$ conidia/g in the wet rice, 10% inoculum size, and use of a polyethylene bag as a container. The polyethylene bag containing inoculated rice was hand-shaken every 12 hr during fermentation. Using optimal conditions, the maximum conidia production obtained was 4.05 g conidia/100 g dry rice after 14 days of cultivation, a rate 2.83 times higher than conidia yield of pre-optimization.

The objective of the experiment was to determine the optimum cultural [moisture levels (55, 60 and 70%), days of fermentation (7, 14 and 21), temperature (25 and $35^{\circ}C$) of incubation)] and nutritional parameters (urea addition (0 and 2%) and variable levels of single super phosphate (0.25 and 0.50% SSP)) for bio-processing of the mustard (Brassica campestris) straw (MS) under solid-state fermentation (SSF) system. The performance of SSF was assessed in terms of favorable changes in cell wall constituents, protein content and in vitro DM digestibility of the MS. Sorghum based inoculum (seed culture) of Ganoderma lucidum to treat the MS was prepared. The 50 g DM of MS taken in autoclavable polypropylene bags was mixed with a pre-calculated amount of water and the particular nutrient in the straw to attained the desired levels of water and nutrient concentration in the substrate. A significant progressive increase in biodegradation of DM (p<0.001), NDF (p<0.01) and ADF (p<0.05) was observed with increasing levels of moisture. Among the cell wall constituents the loss of ADF fraction was greatest compared to that of NDF. The loss of DM increased progressively as the fermentation proceeded and maximum DM losses occurred at 28 days after incubation. The protein content of the treated MS samples increased linearly up to the day $21^{th}$ of the incubation and thereafter declined at day $28^{th}$, whereas the improvement in in vitro DM digestibility were apparent only up to the day $14^{th}$ of the incubation under SSF and there after it declined. The acid detergent lignin (ADL) degradation was slower during the first 7 days of SSF and thereafter increased progressively and maximum ADL losses were observed at the day $28^{th}$ of the SSF. The biodegradation of DM and ADL was not affected by the variation in incubation temperature. Addition of urea was found to have inhibitory effect on fungal growth. The effect of both the levels (0.25 and 0.50) of SSP addition in the substrate, on DM, NDF, ADF, cellulose and ADL biodegradation was similar. Similarly, the protein content and the in vitro DM digestibility remain unaffected affected due to variable levels of the SSP inclusion in the substrate. From the results it may be concluded that the incubation of MS with 60 percent moisture for 21 days at $35^{\circ}C$ with 0.25 percent SSP was most suitable for MS treatment with Ganoderma lucidum. Maximum delignification, enrichment in the protein content and improvement in in vitro DM digestibility were achieved by adopting this protocol of bioprocessing of MS.

During cocoa (Theobroma cacao L.) processing, the accumulated cocoa shell can be used for bioconversion to obtain valuable compounds. Here, we evaluate the effect of solid-state fermentation of cacao flour with Penicillium roqueforti on secondary metabolite composition, phenol, carotenoid, anthocyanin, flavonol, and fatty acids contents, and antioxidant activity. We found that the total concentrations of anthocyanins and flavonols did not change significantly after fermentation and the phenolic compound and total carotenoid concentrations were higher. The fermentation process produced an increase in saponin concentration and antioxidant activity, as well as significant changes in the levels of oleic, linoleic, gamma-linolenic, and saturated fatty acids. Based on our findings, we propose that the reuse of food residues through solid state fermentation is viable and useful.

Solid-state fermentation using hulled barley was carried out to produce enzymes and β-glucan. The one-factor-at-a-time experiments were carried out to determine the optimal composition of the basal medium. The modified synthetic medium composition in liquid-state fermentation was determined to be 70 g/l hulled barley, 0 g/l rice bran, 5 g/l soytone, and 6 g/l ascorbic acid. Optimal pretreatment conditions of hulled barley by solid-state fermentation were evaluated in terms of maximum production of fungal biomass, amylase, protease, and β-glucan, which were 1.26 mg/g, 31310.34 U/g, 2614.95 U/g, and 14.6% (w/w), respectively, at 60 min of pretreatment condition. Thus, the solid-state fermentation process was found to enhance the overall fermentation yields of hulled barley to produce high amounts of enzymes and β-glucan.

밀기울 발효에서 효소회수의 모델로 Aspergillus niger를 고체상태로 발효시켜 조사하였다. 발효시킨 밀기울에서 증류수로 효소추출 효율은 초산 완충액, 구연산 완충액, 구연산-인산 완충액 및 5% 메탄올 처리보다 높았다. 따라서, 추출 용매로 증류수를 이용하여 최적 조건을 상세히 검토하였다. 최적 조건은 고체와 액체 용매를 1:5의 비율로 증류수로서 세 번 세척하였을 때에 최대 회수율을 0.025 U/g으로 확보하였다.

Investigations were carried out on a $\beta$-glucosidase produced by Aspergillus niger under solid-state fermentation conditions as a model of enzyme recovery from fermented wheat bran. The leaching efficiency of distilled water to recover the enzyme from the fermented bran was higher than acetate buffer, citrate buffer, citrate-phosphate buffer and 5% methanol; thus, the conditions were further optimized with distilled water as the extracting agent. After fermented bran was washed three times with distilled water for 1.5 hr each under shaking conditions at 1:5 solid to solvent ratio, a maximum recovery of 0.025 U/g of wheat bran was obtained.

Partial purification of glucoamylase from solid-state fermentation culture was, firstly, investigated by reverse micellar extraction (RME). To avoid back extraction problems, the glucoamylase was kept in the original aqueous phase, while the other undesired proteins/enzymes were moved to the reverse micellar organic phase. The individual and interaction effects of main factors (i.e., pH and NaCl concentration in the aqueous phase, and concentration of sodium bis-2-ethyl-hexyl-sulfosuccinate (AOT) in the organic phase) were studied using response surface methodology. The optimum conditions for the maximum recovery of the enzyme were pH 2.75, 100 mM NaCl, and 200 mM AOT. Furthermore, the optimum organic to aqueous volume ratio ($V_{org}/V_{aq}$) and appropriate number of sequential extraction stages were 2 and 3, respectively. Finally, 60% of the undesired enzymes including proteases and xylanases were removed from the aqueous phase, while 140% of glucoamylase activity was recovered in the aqueous phase and the purification factor of glucoamylase was found to be 3.0-fold.