Improved protease activity of Pixian broad bean paste with cocultivation of Aspergillus oryzae QM-6 and Aspergillus niger QH-3

BACKGROUND: The preparation of broad bean koji is a key process in the production of Pixian broad bean paste (PBP). Protease is essential for the degradation of proteins during PBP fermentation. To obtain broad bean koji with high protease activity using the cocultivated strains of Aspergillus oryzae QM-6 (A. oryzae QM-6) and Aspergillus niger QH-3 (A. niger QH-3), the optimization of acid and neutral protease activities was carried out using Box­Behnken design with response surface methodology (RSM). RESULTS: The optimum conditions were found to be as follows: inoculation proportion (X1), 3:1 (A. oryzae QM-6: A. niger QH-3, w/w); culture temperature (X2), 33°C; inoculum size (X3), 0.5% (w/w); incubation time (X4), 5 d. The acid and neutral protease activities were 605.2 ± 12.4 U/g and 1582.9 ± 23.7 U/g, respectively, which were in good agreement with the predicted values. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the broad bean koji extracellular proteins in the case of cocultivation were richer compared to those in the case of A. oryzae QM-6 or A. niger QH-3 strain only. In addition, the free amino acids (FAAs) in the fermentation product were 55% higher in the cocultivation process than in that involving only A. oryzae QM-6, further confirming the diversity of proteases in the fermentation products. CONCLUSIONS: The optimal conditions of koji-making in PBP were obtained using RSM. The cocultivation of A. oryzae and A. niger increases the overall enzyme activities in the culture medium and the FAAs content, which would thus have potential application in the PBP industry.

Desarrollo y capacidad de remoción de los pesticidas atrazina, clorpirifós y endosulfán por parte de cepas de Aspergillus sección Flavi no toxigénicas / Development and removal ability of non-toxigenic Aspergillus section Flavi in presence of atrazine, chlorpyrifos and endosulfan

This study evaluated the in vitro effect of three concentrations of atrazine, chlorpyrifos and endosulfan on the growth parameters of four non-toxigenic Aspergillus section Flavi strains. The ability of the strains to remove these pesticides in a synthetic medium was also determined. Growth parameters were measured on soil extract solid medium supplied with 5,10 and 20mg/l of each pesticide, and conditioned to -0.70, -2.78, -7.06 and -10.0 water potential (MPa). Removal assays were performed in Czapek Doc medium (CZD) supplied with 20mg/l of each pesticide under optimal environmental conditions (-2.78 of MPa and 25 °C). The residual levels of each pesticide were detected by the reversed-phase HPLC/fluorescence detection system. The lag phases of the strains significantly decreased in the presence of the pesticides with respect to the control media. This result indicates a fast adaptation to the conditions assayed. Similarly, the mycelial growth rates in the different treatments increased depending on pesticide concentrations. Aspergillus oryzae AM 1 and AM 2 strains showed high percentages of atrazine degradation (above 90%), followed by endosulfan (56 and 76%) and chlorpyrifos (50 and 73%) after 30 days of incubation. A significant (p <0.001) correlation (r = 0.974) between removal percentages and growth rate was found. This study shows that non-toxigenic Aspergillus section Flavi strains from agricultural soils are able to effectively grow in the presence of high concentrations of atrazine, chlorpyrifos and endosulfan under a wide range of MPa conditions. Moreover, these strains have the ability to remove high levels of these pesticides in vitro in a short time.

En este estudio se evaluó los efectos in vitro de 3 concentraciones de atrazina, clorpirifós y endosulfán sobre los parámetros de crecimiento de 4 cepas no toxigénicas de Aspergillus sección Flavi. También se evaluó la capacidad de las cepas de remover los pesticidas. Los parámetros de crecimiento se ensayaron en medio agar extracto de suelo suplementado con 5, 10 y 20mg/l de cada pesticida y acondicionado a -0.70, -2.78, -7.06 y -10.0 de potencial de agua (MPa). Los ensayos de remoción se realizaron en medio Czapek Dox con 20mg/l de cada pesticida bajo condiciones óptimas de crecimiento (-2.78 de MPa y 25 °C). Los niveles residuales de atrazina, clorpirifós y endosulfán se detectaron en un sistema HPLC con detección por fluorescencia. La fase de latencia de las cepas disminuyó significantemente en presencia de los pesticidas, indicando una rápida adaptación a dichas condiciones. La velocidad de crecimiento se incrementó considerablemente dependiendo de la concentración de pesticida. Las cepas Aspergillus oryzae AM1 y AM2 mostraron porcentajes elevados de degradación de atrazina (aproximadamente el 90%), seguidos por endosulfán (56 y 76%) y clorpirifós (50 y 73%). Se observó una correlación (r = 0.974) significante (p <0.001) entre el porcentaje de pesticida removido y la velocidad de crecimiento. Este estudio muestra que cepas no-toxigénicas de Aspergillus sección Flavi aisladas de suelos agrícolas desarrollan eficientemente en presencia de altas concentraciones de atrazina, clorpirifós y endosulfán en un amplio rango de MPa. Además, presentan capacidad de remover in vitro altos niveles de pesticidas en corto tiempo.

Heterologous expression and characterization of Aspergillus oryzae acidic protease in Pichia pastoris / 生物工程学报

Acid protease, an important aspartic protease, has been widely used in food, pharmaceutical and tanning industries. To promote the research and application of acid protease, an acid protease gene (pepA) from Aspergillus oryzae was obtained from fermented soy based on metagenome sequencing, and then cloned and transformed into Pichia pastoris GS115 for heterologous expression. The characteristic of recombinant PepA was also investigated. The activity of acid protease in the culture supernatant of P. pastoris was 50.62 U/mL. The molecular mass of PepA was about 50 kDa, and almost no other proteins in the supernatant were observed, as shown by SDS-PAGE. The optimum pH and temperature of PepA were determined as pH 4.5 and 50 ℃. Mn²⁺ and Cu²⁺ enhanced the activity of PepA, whereas Fe³⁺, Fe²⁺ and Ca² had inhibitory effects on its activity. The above findings can provide guidance for heterologous expression and industrial application of acid protease from Aspergillus oryzae.

Construction and application of a novel genetically engineered Aspergillus oryzae for expressing proteases

Background: We aimed to test the possibility of improving polypeptide production from soybean meal fermentation by engineered Aspergillus oryzae strains. Four different protease genes were cloned and transformed into wild-type A. oryzae, and the engineered A. oryzae strains were then used for soybean meal fermentation. Results: The results showed different degrees of improvement in the protease activity of the four transformants when compared with wild-type A. oryzae. A major improvement in the polypeptide yield was achieved when these strains were used in soybean meal fermentation. The polypeptide conversion rate of one of the four transformants, A. oryzae pep, reached 35.9%, which was approximately twofold higher than that exhibited by wild-type A. oryzae. Amino acid content analysis showed that the essential amino acid content and amino acid composition of the fermentation product significantly improved when engineered A. oryzae strains were used for soybean meal fermentation. Conclusions: These findings suggest that cloning of microbial protease genes with good physicochemical properties and expressing them in an ideal host such as A. oryzae is a novel strategy to enhance the value of soybean meal.

Produção de xilanases por fermentação em estado sólido de farelo de trigo utilizando consórcios fúngicos / Production of xylanases by solid state fermentation of wheat bran using fungal consortia

A fim de se agregar valor ao resíduo farelo de trigo gerado em indústrias do setor alimentício avaliou-se, no presente trabalho, o potencial deste subproduto como substrato para produção de enzima xilanase no cultivo em estado sólido, utilizando consórcios fúngicos bem como os fungos Aspergillus oryzae CCT nº 0975 (ATCC9362) eTrichoderma reesei CCT nº 2768 - QM 9414. Para tanto utilizou-se o farelo de trigo, não lavado e não autoclavado, como fonte de carbono e energia na fermentação em estado sólido pelo fungo Aspergillus oryzae que apresentou maior produção do percentual de proteína nas 72 horas de cultivo. Depois de realizado um Delineamento Composto Central Rotacional (DCCR) - planejamento fatorial 23,com três repetições no ponto central e seis pontos axiais - partiu-se para otimização dos fatores que foram considerados significativos no processo: umidade, pH e granulometria. Os fatores foram considerados significativos pela A NOVA com o nível de 95% de confiança e com o resultado otimizado de atividade enzimática de (1.84 ± 0.01) UI/mL utilizando pH 3,3, granulometria de 900,0 µm e umidade de 40%. O caldo enzimático obtido foi considerado eficiente na modificação de tipificação de farinhas de trigo pelo estudo dos parâmetros reológicos do falling number e alveografia sendo estável por cerca de 3 meses

This study aimed to find alternatives for wheat bran disposal destination generated in food industry sector,thus contributing to the reduction of the resultant impact of residue depo-sition in the environment. The poten-tial of the wheat bran as a substrate for xylanase production by solid-state fermentation using fungal con-sortiums as well as Aspergillus ory-zae (ATCC9362) and Trichoderma reesei (2768) was valued. The use of non-washed and non-autoclaved wheat bran as carbon and energy source in solid-state fermentation by A. oryzae fungus showed greater per-centage of produced protein after 72 h of cultivation. The use of a central composite rotatable design(CCRD), 23 factorial planning with three rep-etitions at the central point as well as six axial points, coupled with Sur-face Response Methodology (SRM) allowed to assay the influence of hu-midity, pH, and grain size (indepen-dent variables or factors) on the xy-lanase activity(dependent variable or response) as well as to optimize the best conditions for the enzyme production. The results showed that all factors and their combinations were significant at 95% confidence level. The optimized xylanase activi-ty was (1.84 ± 0.01) UI/mL, obtained at 40% humidity and pH 3.3 with a grain size of 900.0 µm. The produced broth was stable for 3 months and approximately had 50% of the initial xylanase activity at 4°C. SDS-PAGE assay showed that xylanase has 30 kDa molar mass. The obtained en-zymatic broth was efficient to modify wheat flours as shown by the falling number rheologic parameters and alveography assay

Characteristics of Korean Alcoholic Beverages Produced by Using Rice Nuruks Containing Aspergillus oryzae N159-1

Herein, nuruks derived from non-glutinous and glutinous rice inoculated with Aspergillus oryzae N159-1 (having high alpha-amylase and beta-glucosidase activities) were used to produce Korean alcoholic beverages. The resultant beverages had enhanced fruity (ethyl caproate and isoamyl alcohol) and rose (2-phenethyl acetate and phenethyl alcohol) flavors and high taste scores.

Enzyme activity of ß-galactosidase from Kluyveromyces lactis and Aspergillus oryzae on simulated conditions of human gastrointestinal system / Atividade de ß-galactosidase de Kluyveromyces lactis e Aspergillus oryzae, em condições simuladas do sistema gastrintestinal humano

An alternative to relieve the symptoms of lactose intolerance is the intake of the enzyme ?-galactosidase in pharmaceutical dosage forms. The ability of ?-galactosidase produced by Kluyveromyces lactis and Aspergillus oryzae to hydrolyze lactose in simulated conditions of the human gastrointestinal tract was investigated. The experiment was carried out in the optimum temperature for each enzyme activity, 40 and 55°C, respectively, and at the normal human body temperature (37°C) at concentrations of 1.5, 3.0, and 5.0 g/L (enzyme from A. oryzae) or mL/L (enzyme from K. lactis). Both enzymes were completely inactivated under simulated gastric conditions (pH 2). When the enzymes were subjected to simulated small intestine conditions (pH 7.4), lactose hydrolysis has occurred, but at 37°C the percentage was lower than that under the optimal temperatures. At concentrations of 1.5, 3.0, and 5.0 mL/L the enzyme from K. lactis hydrolyzed 76.63%, 88.91% and 94.80% of lactose at 40°C, and 55.99%, 80.91% and 81.53% at 37°C, respectively. In contrast, the enzyme from A. oryzae hydrolyzed 7.11%, 16.18% and 21.29% at 55°C, and 8.4%, 11.85% and 16.43% at 37°C. It was observed that under simulated intestinal conditions, the enzyme from K. lactis was more effective on lactose hydrolysis as compared to the enzyme from A. oryzae. Considering the findings of this study, it is extremely necessary to use an enteric coating on ?-galactosidase capsules so that this enzyme is released only in the small intestine, which is its site of action, thus not suffering the action of the stomach pH.(AU)

Uma das alternativas para amenizar os sintomas da intolerância à lactose é a ingestão de ?-galactosidase em formas farmacêuticas. Neste trabalho avaliou-se a capacidade de hidrólise de ?-galactosidase produzida por Kluyveromyces lactis e Aspergillus oryzae simulando as condições do trato gastrintestinal humano. O teste foi realizado nas temperaturas ótimas de ação para cada enzima, 40 e 55°C, respectivamente, e na temperatura corpórea humana (37°C), nas concentrações de 1,5; 3,0 e 5,0 g/L para a enzima de Aspergillus oryzae ou mL/L para a de Kluyveromyces lactis. Na simulação da condição estomacal humana (pH 2), ambas enzimas foram totalmente inativadas. Quando as enzimas foram submetidas às condições simuladas do intestino delgado (pH 7,4), observou-se hidrólise da lactose, porém, a 37°C, a porcentagem foi menor do que a observada nas temperaturas ótimas de cada enzima. A enzima de K. lactis nas concentrações de 1,5; 3,0 e 5,0 mL/L apresentou hidrólise de 76,63%, 88,91% e 94,80% a 40°C e 55,99%, 80,91% e 81,53%, a 37°C, respectivamente. Nas concentrações 1,5; 3,0 e 5,0 g/L, a porcentagem de hidrólise pela enzima de A. oryzae a 55°C foi de 7,11%, 16,18% e 21,29%. Para esta enzima, nessas concentrações, a hidrólise obtida a 37°C foi 8,4%, 11,85% e de 16,43%. Sob condições intestinais simuladas, a enzima de K. lactis apresentou maior eficiência na hidrólise da lactose quando comparada à enzima de A. oryzae. Considerando-se as etapas avaliadas neste estudo, observa-se que é extremamente necessário o uso de um revestimento entérico em cápsulas de ?-galactosidase, para que esta enzima seja liberada somente no intestino delgado, seu local de ação, não sofrendo, portanto, a ação do pH estomacal.(AU)

Support networks and people with physical disabilities: social inclusion and access to health services / Redes de apoio e pessoas com deficiência física: inserção social e acesso aos serviços de saúde

This study seeks to identify the formation of social support networks of people with physical disabilities, and how these networks can help facilitate access to health services and promote social inclusion. It is a cross-sectional study, with data collected via a form applied to physically disabled persons over eighteen years of age registered with the Family Health Teams of the municipal district of João Pessoa in the state of Paraíba. It was observed that the support networks of these individuals predominantly consist of family members (parents, siblings, children, spouses) and people outside the family (friends and neighbors). However, 50% of the interviewees declared that they could not count on any support from outside the family. It was observed that the support network contributes to access to the services and participation in social groups. However, reduced social inclusion was detected, due to locomotion difficulties, this being the main barrier to social interaction. Among those individuals who began to interact in society, the part played by social support was fundamental.

Este estudo objetiva identificar a constituição das redes de apoio social das pessoas com deficiência física e como estas podem contribuir para facilitar o acesso aos serviços de saúde e a inclusão social das mesmas. Trata-se de um estudo transversal, com dados coletados através de um formulário, aplicado em pessoas com deficiência física maiores de dezoito anos, cadastradas nas Equipes de Saúde da Família do município de João Pessoa (PB). Constatou-se que as redes de apoio dessas pessoas estão constituídas principalmente pelos componentes da dimensão familiar (pais, irmãos, filhos, cônjuges) e extrafamiliar (amigos e vizinhos). No entanto, 50% dos entrevistados relataram não contar com qualquer apoio fora da família. Verificou-se que a rede de apoio contribui para o acesso aos serviços e para a participação em grupos sociais. Evidenciou-se, porém, uma reduzida inserção social, decorrente da dificuldade de locomoção, sendo esta a principal barreira para a interação social. Entre as pessoas que começaram a interagir na sociedade o apoio social foi fundamental.

Insecticidal Activity of Ethyl Acetate Extracts from Culture Filtrates of Mangrove Fungal Endophytes

In the search for novel potent fungi-derived bioactive compounds for bioinsecticide applications, crude ethyl acetate culture filtrate extracts from 110 mangrove fungal endophytes were screened for their toxicity. Toxicity tests of all extracts against brine shrimp (Artemia salina) larvae were performed. The extracts with the highest toxicity were further examined for insecticidal activity against Spodoptera litura larvae and acetylcholinesterase (AChE) inhibition activity. The results showed that the extracts of five isolates exhibited the highest toxicity to brine shrimp at 50% lethal concentration (LC50) values of 7.45 to 10.24 ppm. These five fungal isolates that obtained from Rhizophora mucronata were identified based on sequence data analysis of the internal transcribed spacer region of rDNA as Aspergillus oryzae (strain BPPTCC 6036), Emericella nidulans (strains BPPTCC 6035 and BPPTCC 6038), A. tamarii (strain BPPTCC 6037), and A. versicolor (strain BPPTCC 6039). The mean percentage of S. litura larval mortality following topical application of the five extracts ranged from 16.7% to 43.3%. In the AChE inhibition assay, the inhibition rates of the five extracts ranged from 40.7% to 48.9%, while eserine (positive control) had an inhibition rate of 96.8%, at a concentration of 100 ppm. The extracts used were crude extracts, so their potential as sources of AChE inhibition compounds makes them likely candidates as neurotoxins. The high-performance liquid chromatography profiles of the five extracts differed, indicating variations in their chemical constituents. This study highlights the potential of culture filtrate ethyl acetate extracts of mangrove fungal endophytes as a source of new potential bioactive compounds for bioinsecticide applications.

Aspergillus Associated with Meju, a Fermented Soybean Starting Material for Traditional Soy Sauce and Soybean Paste in Korea

Aspergillus is an important fungal genus used for the fermentation of Asian foods; this genus is referred to as koji mold in Japan and China. A. oryzae, A. sojae, and A. tamari are used in the production of miso and shoyu in Japan, but a comprehensive taxonomic study of Aspergillus isolated from Meju, a fermented soybean starting material for traditional soy sauce and soybean paste in Korea, has not been conducted. In this study, various Aspergillus species were isolated during a study of the mycobiota of Meju, and the aspergilli were identified based on phenotypic characteristics and sequencing of the beta-tubulin gene. Most strains of Aspergillus were found to belong to the following sections: Aspergillus (n = 220), Flavi (n = 213), and Nigri (n = 54). The most commonly identified species were A. oryzae (n = 183), A. pseudoglaucus (Eurotium repens) (n = 81), A. chevalieri (E. chevalieri) (n = 62), A. montevidensis (E. amstelodami) (n = 34), A. niger (n = 21), A. tamari (n = 15), A. ruber (E. rubrum) (n = 15), A. proliferans (n = 14), and A. luchuensis (n = 14); 25 species were identified from 533 Aspergillus strains. Aspergillus strains were mainly found during the high temperature fermentation period in the later steps of Meju fermentation.

The Mycobiota of Air Inside and Outside the Meju Fermentation Room and the Origin of Meju Fungi

The fungi on Meju are known to play an important role as degrader of macromolecule of soybeans. In order to elucidate the origin of fungi on traditional Meju, mycobiota of the air both inside and outside traditional Meju fermentation rooms was examined. From 11 samples of air collected from inside and outside of 7 Meju fermentation rooms, 37 genera and 90 species of fungi were identified. In outside air of the fermentation room, Cladosporium sp. and Cladosporium cladosporioides were the dominant species, followed by Cladosporium tenuissimum, Eurotium sp., Phoma sp., Sistotrema brinkmannii, Alternaria sp., Aspergillus fumigatus, Schizophyllum commune, and Penicillium glabrum. In inside air of the fermentation room, Cladosporium sp., Aspergillus oryzae, Penicillium chrysogenum, Asp. nidulans, Aspergillus sp., Cla. cladosporioides, Eurotium sp., Penicillium sp., Cla. tenuissimum, Asp. niger, Eur. herbariorum, Asp. sydowii, and Eur. repens were collected with high frequency. The concentrations of the genera Aspergillus, Eurotium, and Penicillium were significantly higher in inside air than outside air. From this result and those of previous reports, the origin of fungi present on Meju was inferred. Of the dominant fungal species present on Meju, Lichtheimia ramosa, Mucor circinelloides, Mucor racemosus, and Scopulariopsis brevicaulis are thought to be originated from outside air, because these species are not or are rarely isolated from rice straw and soybean; however, they were detected outside air of fermentation room and are species commonly found in indoor environments. However, Asp. oryzae, Pen. polonicum, Eur. repens, Pen. solitum, and Eur. chevalieri, which are frequently found on Meju, are common in rice straw and could be transferred from rice straw to Meju. The fungi grow and produce abundant spores during Meju fermentation, and after the spores accumulate in the air of fermentation room, they could influence mycobiota of Meju fermentation in the following year. This could explain why concentrations of the genera Aspergillus, Eurotium, and Penicillium are much higher inside than outside of the fermentation rooms.

Soluble extract of soybean fermented with Aspergillus oryzae GB107 inhibits fat accumulation in cultured 3T3-L1 adipocytes

BACKGROUND/OBJECTIVES: This study was conducted to investigate the effects of fermented soybean (FS) extract on adipocyte differentiation and fat accumulation using cultured 3T3-L1 adipocytes. MATERIALS/METHODS: 3T3-L1 adipocytes were treated with FS and nonfermented soybean (NFS) extract during differentiation for 10 days in vitro. Oil red O staining was performed and glycerol-3-phosphate dehydrogenase (GPDH) activity was measured for analysis of fat accumulation. Expressions of adipogenic genes were measured. RESULTS: Soluble extract of soybean fermented with Aspergillus oryzae GB107 contained higher levels of low-molecular-weight protein than conventional soybean protein did. FS extract (50 microg/ml) inhibited adipocyte differentiation and fat accumulation during differentiation of 3T3-L1 preadipocytes for 10 days in vitro. Significantly lower GPDH activity was observed in differentiated adipocytes treated with the FS extract than those treated with NFS extract. Treatment with FS extract resulted in decreased expression levels of leptin, adiponectin, and adipogenin genes, which are associated with adipogenesis. CONCLUSIONS: This report is the first to demonstrate that the water-soluble extract from FS inhibits fat accumulation and lipid storage in 3T3-L1 adipocytes. Thus, the soybean extract fermented with A. oryzae GB107 could be used to control lipid accumulation in adipocytes.

Antioxidant activity and anti-inflammatory activity of ethanol extract and fractions of Doenjang in LPS-stimulated RAW 264.7 macrophages

BACKGROUND/OBJECTIVES: Fermentation can increase functional compounds in fermented soybean products, thereby improving antioxidant and/or anti-inflammatory activities. We investigated the changes in the contents of phenolics and isoflavones, antioxidant activity and anti-inflammatory activity of Doenjang during fermentation and aging. MATERIALS/METHODS: Doenjang was made by inoculating Aspergillus oryzae and Bacillus licheniformis in soybeans, fermenting and aging for 1, 3, 6, 8, and 12 months (D1, D3, D6, D8, and D12). Doenjang was extracted using ethanol, and sequentially fractioned by hexane, dichloromethane (DM), ethylacetate (EA), n-butanol, and water. The contents of total phenolics, flavonoids and isoflavones, 2,2-diphenyl-1 picryl hydrazyl (DPPH) radical scavenging activity, and ferric reducing antioxidant power (FRAP) were measured. Anti-inflammatory effects in terms of nitric oxide (NO), prostaglandin (PG) E2 and pro-inflammatory cytokine production and inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions were also measured using LPS-treated RAW 264.7 macrophages. RESULTS: Total phenolic and flavonoid contents showed a gradual increase during fermentation and 6 months of aging and were sustained thereafter. DPPH radical scavenging activity and FRAP were increased by fermentation. FRAP was further increased by aging, but DPPH radical scavenging activity was not. Total isoflavone and glycoside contents decreased during fermentation and the aging process, while aglycone content and its proportion increased up to 3 or 6 months of aging and then showed a slow decrease. DM and EA fractions of Doenjang showed much higher total phenolic and flavonoid contents, and DPPH radical scavenging activity than the others. At 100 microg/mL, DM and EA fractions of D12 showed strongly suppressed NO production to 55.6% and 52.5% of control, respectively, and PGE2 production to 25.0% and 28.3% of control with inhibition of iNOS or COX-2 protein expression in macrophages. CONCLUSIONS: Twelve month-aged Doenjang has potent antioxidant and anti-inflammatory activities with high levels of phenolics and isoflavone aglycones, and can be used as a beneficial food for human health.

Purification and Partial Characterization of a Non-specific Acid Phosphatase Degrading NAD from Aspergillus oryzae NRRL447.

A non specific acid phosphatase from Aspergillus oryzae NRRL447 catalyzes the phosphate hydrolysis from nicotinamide adenine dinucleotide forming nicotinamide riboside, adenosine and Pi as the final products of the reaction. The enzyme was purified to homogeneity by a sequential treatment of acetone fractionation, DEAE-cellulose chromatography and gel filtration chromatography. The enzyme was purified 400-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 52 kDa. The enzyme displayed maximum activity at pH 5.0 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg2+, Ca2+ whereas inhibited strongly by F-, Mo04 -, Cu2+ and Fe2+. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The substrate concentration-activity relationship is the hyperbolic type and the apparent Km for NAD+ was 6.25 x 10-4 M.

Gene cloning, expression of a feruloyl esterase A and purification of its hydrolysis products / 生物工程学报

To express feruloyl esterase A from Aspergillus oryzae in Pichia pastoris expression system and study its hydrolysis function, explore the conditions and effects of purification for ferulic acid extracts by macroporos resin. Using the total RNA from A. oryzae CICC 40186 as the template, we amplified coding sequence AorfaeA encoding a mature feruloyl esterase A (AorFaeA) by RT-PCR technique. Then, the coding sequence AorfaeA was successfully expressed in Pichia pastoris GS115 mediated by an expression plasmid pPIC9K. The purified recombinant AorFaeA (reAorFaeA) showed one single band on SDS-PAGE with an apparent molecular weight of 39.0 kDa. The maximum activity of reAorFaeA to methyl ferulate, measured by high-performance liquid chromatography (HPLC), was 58.35 U/mg. Then, reAorFaeA was used to release ferulic acid from de-starched wheat bran in the presence of xylanase. The purification tests for ferulic acid from the enzymatic hydrolysate were carried out with preselected macroporous resins. The results showed that macroporous resin HPD-300 had much higher adsorption and desorption capacities. Ferulic acid could be quantitatively recovered by 50% of the eluent concentration at a flow speed of 1 mL/min. Under the purification condition, the recovery ratio of ferulic acid was 92%, and the content of ferulic acid was increased from 0.13% in the raw material to 10.55%. This work exploits the breakdown of ferulic acid by recombinant enzymeand provids a good strategy to its "green production".

Aspergillus oryzae S-03 Produces Gingipain Inhibitors as a Virulence Factor for Porphyromonas gingivalis

Oral infection with Porphyromonas (P.) gingivalis causes periodontitis that is manifested by the destruction of gingival connective tissues. Although a few types of antibiotics are effective against the infection, its use induces the appearance of drug-resistant bacteria. The present study shows that the fermented product of Aspergillus (A.) oryzae S-03, cultivated on the fat-removed soybean, inhibits the cell growth of the P. gingivalis. Likewise, the fermented product of the S-03 strain cultured for 26~42 h displays an inhibitory activity to gingipain as a virulence factor of P. gingivalis. The activity is not lost even with heat treatment at 100degrees C for 15 min. We also demonstrate that the S-03 strain exhibits high protease activity. In addition, the strain does not produce aflatoxin because of the loss of a regulatory gene, aflR, necessary for the toxin biosynthesis.

Occupational asthma induced by beat-galactosidase

Galactosidase is generated from Aspergillus oryzae, which is widely used for antidiarrhea medicine to infants. Antibiotics and digestives were reported as a causative allergen inducing occupational asthma. Galatosidase-induced occupational asthma has not been reported yet. A forty-year-old female has suffered from rhinorrhea, sneezing, and nasal obstruction 1 year after handling galactosidase at obstetric and pediatric hospital, and then dyspnea appeared later. Skin prick test with inhalent allergens, beta-galactosidase, and Aspergillus oryzae showed strong positive reaction to beta-galactosidase only. Immunoinhibition test with beta-galactosidase and A. oryzae revealed inhibition to beta-galactosidase only. Bronchial provocation test with beta-galactosidase showed the dual asthmatic response. With these results, we confirmed that the patient has beta-galactosidase-induced occupational asthma and rhinitis.

Exploration of optimum conditions for production of saccharogenic mixed grain beverages and assessment of anti-diabetic activity / 한국영양학회지

PURPOSE: This study was conducted to establish the production conditions through optimization of the production process of beverages using Aspergillus oryzae CF1001, and to analyze volatile compounds and antidiabetic activity. METHODS: The optimum condition was selected using the response surface methodology (RSM), through a regression analysis with the following independent variables gelatinization temperature (GT, X1), saccharogenic time (ST, X2), and dependent variable; DeltaE value (y). The condition with the lowest DeltaE value occurred with combined 45 min ST and 50degrees C GT. The volatile compounds were analyzed quantitatively by GC-MS. RESULTS: Assessment of antidiabetic activity of saccharogenic mixed grain beverage (SMGB) was determined by measurement of alpha-glucosidase inhibition activity, and glucose uptake activity and glucose metabolic protein expression by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. Results of volatile compounds analysis, 62 kinds of volatile compounds were detected in SMGB. Palmitic acid (9.534% ratio), benzaldehyde (8.948% ratio), benzyl ethyl ether (8.792% ratio), ethyl alcohol (8.35% ratio), and 2-amyl furan (4.826% ratio) were abundant in SMGB. We confirmed that alpha-glucosidase inhibition activity, glucose uptake activity, and glucose-metabolic proteins were upregulated by SMGB treatment with concentration dependent manner. CONCLUSION: Saccharogenic mixed grain beverage (SMGB) showed potential antidiabetic activity. Further studies will be needed in order to improve the taste and functionality of SMGB.

Effect of fermented soybean curd residue (FSCR; SCR-meju) by aspergillus oryzae on the anti-obesity and lipids improvement / 한국영양학회지

In this study, we designed to confirm the dietary effect of anti-obesity of fermented soybean curd residue (FSCR; SCR-Meju; Biji-meju) by A. oryzae, which is well known as a Korean traditional meju microbe. We observed that body weight gain, serum and hepatic lipid profile, as well as the activity of ROS generating enzyme and ROS scavenging enzyme in high-fat diet induced obese mice fed experimental diet (SCR and SCR-meju). Body weight gain and epididymal fat weight of HC (high-fat diet control) was markedly higher than that of NC (Normal control). Conversely, body weight gain and epididymal fat weight of the SCR (Biji) and SCR-meju (Biji-meju) group was significantly lower than that of HC; these of the SCR-meju group was lower than that of the SCR group. Furthermore, serum TG and total-cholesterol and LDL-cholesterol contents of SCR and SCR-meju groups were lower than that of HC, and HDL-cholesterol level of the SCR-meju group was significantly higher than that of HC. In conclusion, although precise mechanisms of the antiobese effects of SCR-meju in this study are unknown, the present study provides an experimental evidence that SCR-meju may prevent obesity and obesity related metabolic syndromes, such as hyperlipidemia, hypertension and diabetes, and liver disease by high-fat diet. Nevertheless, further study in this filed will be needed.