Characterisation of acid protease expressed from Aspergillus oryzae MTCC 5341

Aspergillus oryzae (MTCC 5341) has the largest expanse of hydrolytic genes, that includes 135 protease genes coding for alkaline, acid as well as neutral proteases. This study reports the purification and characterisation of an acid protease obtained from A. oryzae MTCC 5341. A. oryzae MTCC 5341 produces one of the highest reported acid protease activities reported so far (8.3 × 10⁵ U/g dry bran). The extracellular acid protease (47 kDa) was found to be active in the pH range 3.0–4.0 and stable in the pH range 2.5–6.5. Optimum temperature for activity was 55 °C. The protease was purified 17–fold with a yield of 29%. The enzyme was characterised to be an aspartate protease by inhibition studies, using pepstatin and its ability to activate trypsinogen. The enzyme cleaved the B-chain of insulin at L–V and Y–T residues.     

Optimisation of antioxidants extraction from soybeans fermented by Aspergillus oryzae

The extraction of antioxidant compounds from soybeans fermented with Aspergillusoryzae was optimised using a factorial design. A kinetic study of the total phenolic production and DPPH radical scavenging activity was first performed at the points selected in the factorial design. In both cases, the experimental profiles were fitted to a modified first-order kinetic model. To investigate the combined effects of temperature and solvent concentration on the extraction, the parameters obtained from the fitted kinetic models were used as response variables in a rotatable second-order design with quintuple replications in the centre of the experimental domain. The results obtained indicate that temperature had the most significant effect. The response surfaces show a maximum in the experimental domain studied. The optimum conditions for the extraction of total phenolic content were 65.3 °C and 73.1% ethanol, in which 56.2 mg of GAE/g were predicted. A scavenging activity of 81.6% DPPH radical was predicted at the optimum conditions of 61.6 °C and 60% ethanol.     

Galacto-oligosaccharide synthesis by immobilized Aspergillus oryzae β-galactosidase

Aspergillus oryzae β-galactosidase was immobilized by three different techniques, namely adsorption on celite, covalent coupling to chitosan and aggregation by cross-linking. These techniques were compared in terms of the yield of immobilized preparation, enzymatic characteristics, stability and efficiency in oligosaccharide synthesis. Immobilization led to increase in K_m in each case. Immobilization on chitosan gave maximum enzyme yield and oligosaccharide synthesis. At 60 °C, the chitosan-immobilized enzyme was stabilized (by 1.6-fold) due to protection effect of the matrix. However, at 65 °C, the t_1/2 of cross-linked enzyme aggregates (CLEA) of β-galactosidase was 1.07 h as compared to 0.79 h in the case of free enzyme. Both chitosan-immobilized enzyme and CLEA were used for oligosaccharide synthesis. Using 20% (w/v) lactose, the chitosan-immobilized enzyme gave maximum oligosaccharide yield (17.3% of the total sugar) as compared to free enzyme (10.0%) in 2 h at 40 °C. CLEA were instead found effective in lactose hydrolysis yielding 78% monosaccharide in 12 h.     

Identification and characterization of a novel fermented substance produced by edible Aspergillus oryzae AO-1 that inhibits DPP-IV activity

We identified a novel fermented substance (FKA) produced by the edible Aspergillus oryzae strain AO-1 that inhibited dipeptidyl peptidase IV (DPP-IV) (IC₅₀ = 3.41 mg·mL^− 1). HPLC analysis of FKA showed specific one metabolite (WYK-1), which inhibited DPP-IV (IC₅₀ = 6.98 μM). WYK-1 was identified as a tetrahydroxyisoquinoline derivative. Interestingly, we examined 60 strains of A. oryzae, AO-1 was the only A. oryzae strain that produces WYK-1. This is the first report that an edible A. oryzae strain produces a DPP-IV inhibitor such as WYK-1. This study also suggests that FKA has applications in the development of novel antihyperglycemic therapeutics or functional foods.     

Galacto-oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol

The synthesis of galacto-oligosaccharides (GOS) by the action of Aspergillus oryzae β-galactosidase free and immobilized on magnetic polysiloxane-polyvinyl alcohol (mPOS-PVA) was studied. A maximum GOS concentration of 26% (w/v) of total sugars was achieved at near 55% lactose conversion from 50%, w/v lactose solution at pH 4.5 and 40 °C. Trisaccharides accounted for more than 81% of the total GOS produced. GOS formation was not considerably affected by pH and temperature. The concentrations of glucose and galactose encountered near maximum GOS concentration greatly inhibited the reactions and reduced GOS yield. GOS formation was not affected by enzyme immobilization in the mPOS-PVA matrix, indicating the absence of diffusional limitations in the enzyme carrier. Furthermore, this water insoluble magnetic derivative was reutilized 10-times and retained about 84% of the initial activity. In addition, the kinetic parameters for various initial lactose concentrations were determined and compared for the free and immobilized enzyme.     

Comparison of hydrolysis characteristics on defatted peanut meal proteins between a protease extract from Aspergillus oryzae and commercial proteases

A crude protease extract (CPE) was prepared from Aspergillus oryzae HN 3.042 in this work. Three commercial proteases (Alcalase 2.4L, Protamex and papain) and CPE were employed to hydrolyse defatted peanut meal (DPM). CPE was found to be the most effective protease with protein recovery of 80.6%. Moreover, CPE produced a higher degree of hydrolysis (DH, 43.4%) than the tested commercial proteases. The test of molecular weight distribution indicated that DPM proteins were mainly consisted of >10 KDa fraction (86.6%), whereas 3–6 KDa fraction was observed to be the main fraction of all the hydrolysates. CPE hydrolysate possessed a higher nutritional quality than DPM and other hydrolysates on the basis of FAO/WHO (1991) reference pattern. The sensory taste evaluation showed that CPE hydrolysate had better taste than other hydrolysates.     

Effects of hypobaric and temperature-dependent storage on headspace aroma-active volatiles in common squid miso

The purpose of this study was to elucidate the effects of storage at hypobaric (10 kPa) atmosphere at room temperature (25 °C) and at a low temperature of 10 °C at atmospheric pressure on the headspace volatiles of miso prepared from common squid meat during 270 days of storage. Based on the odor active values of the volatiles detected, 2-methylpropanal, 3-methylbutanal, 3-methyl-1-butanol, n-ethyl decanoate, 2,3-butanedione, dimethyl disulfide, methional, and 2-methyl butanoic acid were identified as key aroma compounds in squid miso. Low-temperature storage appeared to retard volatile compound formation and extent the shelf life. Hypobaric storage induced a significant reduction in lipid oxidation products, particularly aldehydes and ketones. The contents of sulfur-containing compounds and acids were significantly low; however, esters had relatively higher levels in hypobaric conditions. Production of furans and their derivatives were also found to be controlled by hypobaric storage. Therefore, hypobaric storage can be considered as an effective means of preserving squid miso and related fish paste products to prolong shelf-life in order to maintain aroma attributes.     

Advantages of a proteolytic extract by Aspergillus oryzae from fish flour over a commercial proteolytic preparation

Proteolytic extract (CPE) was produced by Aspergillus oryzae 2095 under solid state fermentation using a mix of fish flour with polyurethane foam (70:30, w/w) of particle size of 0.5 mm. The proteolytic activity from CPE was compared to a commercial protease (Flavourzyme 500 MG^®). The maximal activity for both extracts was found at pH 8 and 50 °C. The half-lives of CPE and Flavourzyme 500 MG^® were 52 and 25 min at 50 °C, respectively. Furthermore, the hydrolytic activity for both extracts was tested on muscle of giant sea bass (Epinephelus morio), CPE produced a higher degree of hydrolysis (DH) than Flavourzyme 500 MG^® (22.2 and 9.1%, respectively) under optimal experimental conditions for each extract.     

Production and analysis of ochratoxin A produced by Aspergillus ochraceus ITEM 5137 in submerged culture

A rapid simple and economical method was described for the determination of ochratoxin A produced by Aspergillusochraceus ITEM 5117 grown in a biofermenter in submerged culture. The ochratoxin A was determinate with RP-HPLC-FLD (reverse phase high performance liquid chromatography) with direct injection in the HPLC apparatus using a C18 column. The mycotoxin was completely resolved by using the mixture acetonitrile, water and acetic acid (49:49:2 v/v) as the mobile phase with a flow rate of 1.0 mL/min. Mean recoveries of ochratoxin A ranged from 95.36% to 103.15%. The limit of detection for ochratoxin A in medium was found to be 1 μg L⁻¹.     

Evaluation of monosodium glutamate,disodium inosinate and guanylate umami taste by an electronic tongue

An electronic tongue (e-Tongue) was used to evaluate umami taste and compare different flavor enhancers (concentration and manufacture). Monosodium glutamate and three kinds of disodium inosinate and guanylate (I + G) were analyzed. The obtained data were analyzed by principal component analysis (PCA). The first principal component represented the intensity of umami taste. Based on Euclidean distance between ‘I + G’ samples and MSG (0.5%) on the PCA score plot, the umami intensity of all samples was ranked. The ‘I + G’ samples produced by three manufactures were successfully discriminated on the second principal component with a high discrimination index (82.26). Besides, the drift phenomenon of sensor was discussed and finishing sensor analyses in a continuous time could help avoid the drift. This study showed that e-Tongue was useful in analyzing the umami taste and might be a complementary tool in evaluation of flavor enhancer.     

Cloning of Aspergillus oryzae Aovps5 gene,homologous to vacuolar protein sorting associated gene VPS5 and construction of the disruptant

Aovps5 gene was isolated from Aspergillus oryzae as a homologue to S. cerevisiae VPS5 gene which encodes a polypeptide consisting of 451 amino acids that is nearly 32% homologous to Vps5p. Three Aovps5 gene disruptants were generated and they showed higher activity of tripeptidyl peptidase, which is mainly detected in vacuoles, in their culture medium. Higher amount of nitrogenous constituent was found in the filtrate of wheat gluten degraded by addition of culture medium of these disruptants than that of wild type strain. These results suggest that disruption of Aovps5 may contribute to production of fermented foods.     

Grain loss caused by Tribolium castaneum, Sitophilus oryzae and Acanthoscelides obtectus in stored durum wheat and beans treated with Beauveria bassiana

The effect of the fungal entomopathogen Beauveria bassiana (Balsamo) Vuillemin (Hyphomycete) on the losses caused to durum wheat and beans by storage insects was investigated. Grains were infested with Tribolium castaneum (Herbst), Sitophilus oryzae (L.) and Acanthoscelides obtectus (Say). Beauveria bassiana was produced in inoculated autoclaved rice. The spore formulation (ground rice and B. bassiana) was applied to grain (wheat or bean) and shaken to evenly cover the grain. Adults of T. castaneum or S. oryzae were added to wheat and adults of A. obtectus to bean. Five replicates were set up for each treatment and controls (milled rice but no conidia). The insecticidal effect of B. bassiana was tested by measuring the fresh weight and weight loss of grains after four months of storage. Wheat grains infested with S. oryzae without the conidia was significantly more damaged by weevils than grains treated with B. bassiana. The mean fresh weight of grains with the conidia was significantly greater (18.4%) than the corresponding mean without the fungus when S. oryzae were present. Percentage weight loss decreased by 81.5% and was significantly smaller than the loss from the untreated grain. Significant differences were not found in the fresh weight of seed infested with T. castaneum or A. obtectus in treated or untreated grain nor in the percentage weight loss of grains infested with these insects, with and without B. bassiana.     

Survival and reproduction of Tribolium castaneum (Herbst), Rhyzopertha dominica (F.) and Sitophilus oryzae (L.) following periods of starvation

This study determined the starvation tolerance of Tribolium castaneum (Herbst), Rhyzopertha dominica (F.) and Sitophilus oryzae (L.) in terms of both adult survival and reproduction, the impact of starvation on reproduction not having been studied before. Experiments were conducted at 30 °C and 55% or 70% r.h. using a laboratory strain and a field strain of each species. The number of progeny was a better indicator of the impact of starvation on a species than adult survival. Tribolium castaneum was the most tolerant species, requiring up to 35 d starvation before no progeny were produced. Rhyzopertha dominica and S. oryzae required up to 8 d starvation before no progeny were produced. The results suggest that hygiene will have a greater impact on populations of S. oryzae and R. dominica than T. castaneum.     

DNA identification of two laboratory colonies of the weevils, Sitophilus oryzae (L.) and S. zeamais Motschulsky (Coleoptera: Curculionidae) in Taiwan

DNA amplification fingerprinting (DAF) and nuclear ribosomal DNA (nrDNA) amplification were used to discriminate between two laboratory colonies of two closely related species of weevils: the rice weevil, Sitophilus oryzae (L.), and the maize weevil, S. zeamais Motschulsky. For DAF, three sets of primers (aldolase, prolactin receptor, and interleukin-1β) were used for identification by polymerase chain reaction (PCR) and agarose gel electrophoresis, and the highly similar patterns of the resultant amplicons reconfirmed that the two weevils are closely related. The fragments of nrDNA amplification showed that for S. oryzae and S. zeamais, the homologies of the nucleotide sequences of the internal transcribed spacer regions, ITS-1 and ITS-2, were unusually high, at 96% and 97%, respectively. Based on the ITS-1 and ITS-2 sequences, two species-specific primer sets were designed: with the primer set ITS3/So, the predicted 450 bp DNA fragment was yielded with S. oryzae genomic DNA after PCR amplification (n=10), but no PCR product was obtained with S. zeamais (n=10); with the primer set ITS3/Sz, the same 10 S. zeamais specimens yielded a 550 bp DNA fragment, but S. oryzae yielded no amplicons. In view of the difficulty of distinguishing between these two closely related species, the specificity and availability of these two primer sets might prove to be a useful tool for distinguishing between them. However, the nrDNA sequences of the ITS-1 and ITS-2 regions of geographically isolated populations of both weevils still need to be elucidated, and the applicability of this technique to different geographical populations will need to be confirmed by further study.     

Inhibitory effect of the essential oil of Curcuma longa L. and curcumin on aflatoxin production by Aspergillus flavus Link

Aflatoxins are highly toxic, mutagenic, teratogenic and carcinogenic mycotoxins. Consumption of aflatoxin-contaminated food and commodities poses serious hazards to the health of humans and animals. Turmeric, Curcuma longa L., is a native plant of Southeast Asia and has antimicrobial, antioxidant and antifungal properties. This paper reports the antiaflatoxigenic activities of the essential oil of C. longa and curcumin. The medium tests were prepared with the oil of C. longa, and the curcumin standard at concentrations varied from 0.01% to 5.0%. All doses of the essential oil of the plant and the curcumin standard interfered with mycotoxin production. Both the essential oil and curcumin significantly inhibited the production of aflatoxins; the 0.5% level had a greater than 96% inhibitory effect. The levels of aflatoxin B₁ (AFB₁) production were 1.0 and 42.7 μg/mL, respectively, for the samples treated with the essential oil of C. longa L. and curcumin at a concentration of 0.5%.