Efficacy of supplementing Aspergillus awamori in enhancing growth performance, gut microbiota, digestibility, immunity, and antioxidant activity of heat-stressed broiler chickens fed diets containing olive pulp.

BACKGROUND: Gut microbes play a significant role in digestion, developing immunity, and intestinal health. Therefore, direct-fed microbials are used to modify gut microbiota, maintain a healthy digestive system, enhance immunity, and promote the broilers' performance. In addition, it has a role in improving the utilization of unconventional feed ingredients (olive pulp, OP). This study provides the potential role of Aspergillus awamori in enhancing gut microbial content, nutrient utilization, growth performance, and antioxidative status in heat-stressed broiler chickens fed diets containing olive pulp. METHODS: Three hundred chicks (Ross 308; one day old) were divided into four treatment groups (75 chick/ group) randomly, as follows; CON: chicks fed a basal diet based on corn and soybean meal, OP10: chicks fed a diet containing 10% OP, OA1: chicks fed a diet containing OP with A. awamori at 100 mg per kg, OA2: chicks fed a diet containing OP with A. awamori at 200 mg per kg. RESULTS: Adding A. awamori to the broiler diet that contains OP had a positive effect on productive performance via enhancing nutrition digestibility, body weight gain, feed conversion ratio, and carcass characteristics. A. awamori supplementation had a positive impact on immune responses by increasing serum immunoglobulin G and the relative weight of bursa of Fabricius (P < 0.05) compared to the other groups. Chickens fed A. awamori showed a noticeable improvement in the oxidative status through the increase in the level of serum superoxide dismutase, and glutathione peroxidase, and the decrease in the level of malondialdehyde. Feeding A. awamori also modified the intestinal microbial content by increasing the population of Lactobacillus (P < 0.05). CONCLUSIONS: Our study indicated that adding 200 mg A. awamori reduced the negative effect of heat stress by modifying the microbial content of the intestine, immune response, and enhancing feed utilization, thus improving broiler performance, as well as, improving the nutritional value of the olive pulp. Therefore, adding A. awamori to the OP diet can be effectively used in heat-stressed broiler diets.

First Report of Root Rot on Rhizome of Polygonatum kingianum Caused by Aspergillus awamori.

Polygonatum kingianum Coll. et Hemsl (Huangjing), which belongs to the family Asparagaceae, is a perennial traditional Chinese herb with homologous medicinal and edible value (Liu et al., 2021). Huangjing is known to promote blood circulation; it has anti-inflammatory properties, increases immunity, and provides hypoglycemic treatments (Ma et al., 2019). Root rot-infected P. kingianum exhibited withering yellow leaves and stems, rhizome rot, slowed growth, and plant death. In recent years, with an average incidence of up to 45%, the spread of HJ root rot (rhizome and stem bases) has resulted in a significant reduction in the quality and up to 63% reduction in the yields of Sichuan Junlian (104.5°E, 28.2°N) and Guizhou Zhunyi (107.0°E, 27.7°N). After collecting the diseased samples, we used the tissue isolation method to isolate the pathogenic fungi (Wu et al., 2020). Four fungal isolates associated with root rot were obtained: HJ-G2 (two strains), HJ-G3 (one strain), HJ-G4 (one strain), and HJ-G6 (two strains), of which HJ-G2 and HJ-G6 were the dominant species. To determine pathogenicity of each strain, tests were conducted by wounding rhizomes wth an inoculation needle and the pathogen strain was inoculated onto the wound and symptoms observed. The results reveal that HJ-G6 exhibited the strongest pathogenicity against P. kingianum (Figure 1). The HJ-G6 colonies were black, grew rapidly, and produced a large number of spores (Figure 1). A spherical apical sac (conidial head) is formed at the top with two palisades of cells, metulae and phialides, which are shaped radially and produce a large number of spores with 2-5 um in diameter (Figure 2). Morphological observations revealed that the isolate was consistent with Aspergillus awamori (Naher et al., 2021). To further confirm the fungal species, the ribosomal internal transcribed spacer (ITS), ß-tubulin (TUB), and elongation factor 1-alpha (EF-1a) gene regions were amplified with ITS1/ITS4, Bt2a/ Bt2b, and EF1/EF2. Primer and PCR amplification were performed as previously described (Paul et al., 2017). The sequences were compared with those obtained from GenBank. The ITS sequences (GenBank accession number OR682143) of the isolates (HJ-G6) were 100% identical to those of the strain PANCOM10 (GenBank accession number MT007535.1) of Aspergillus awamori. The EF-1a sequences (GenBank accession OR752352) of the isolates (HJ-G6) were 98% identical with strain ITEM 4777 (GenBank accession FN665402.1) of Aspergillus awamori. The TUB sequences (GenBank accession number OR752351) of the isolate (HJ-G6) were 100% identical with strain AF158 (GenBank accession MH781275.1) of Aspergillus awamori. Three maximum likelihood trees were constructed using MEGA v5.0 (Kumar et al., 2018) based on the sequences (ITS, TUB, and EF-1a) of the HJ-G6 strain and that of Aspergillus spp. previously deposited in GenBank (Paul et al., 2017). Phylogenetic analysis showed that HJ-G6 belonged to the Aspergillus awamori clade (Figure 3). Combined with morphological analysis and DNA sequencing, HJ-G6 was identified as Aspergillus awamori. To verify pathogenicity, P. kingianum roots were inoculated with the colonized agar discs of the isolates. P. kingianum plants inoculated with uncolonized agar discs were used as controls. After inoculation, P. kingianum roots were moved to the inoculation chamber under high humidity at 28 °C for 1 d and then transferred to a greenhouse. Previous studies have reported that Fusarium sp. are root rot pathogens in the rhizomes of medicinal plants (Pang et al., 2022; Song et al., 2023). In this study, HJ-G2, HJ-G3, and HJ-G4 were used as the positive controls. Typical symptoms of root rot appeared 3 days after inoculation and were similar to those observed in the field, whereas the control plants remained symptomless. According to the results of the inoculation experiment, the pathogenicity of Aspergillus awamori to P. kingianum root rot was significantly stronger than that of Fusarium (Figure 1). The pathogen was isolated from the rotting root of P. kingianum and the ITS region was sequenced again. Alignment analysis of the ITS sequences revealed that the causal agents were consistent with those of the original isolates. These studies fulfill Koch's postulates. As far as we know, this is the first report of Aspergillus awamori causing root rot in P. kingianum.

Aspergillus awamori: potential antioxidant, anti-inflammatory, and anti-apoptotic activities in acetic acid-induced ulcerative colitis in rats.

Ulcerative colitis (UC) is a chronic colonic inflammation with a significant health hazard. Aspergillus awamori (A. awamori) is a microorganism with various bioactive compounds with natural antioxidant and anti-inflammatory properties. The present work aimed to elucidate the protective and therapeutic effects of varying concentrations of A. awamori against acetic acid (AA)-induced ulcerative colitis (UC) in rats. Nine groups of albino male rats were established: a control negative group (G1), a control positive group (G2,AA), and preventive protocol groups (including G3A, G4A, and G5A) that received 100 mg, 50 mg, and 25 mg/kg b.w, respectively, of A. awamori orally and daily from the 1st day of the experiment and for 7 consecutive days. Then, they were subjected to one dose of AA intrarectally on day 8th. G3B, G4B, and G5B were termed as curative protocol groups that received one dose of AA on day 8th and then administered 100 mg, 50 mg, and 25 mg/kg b.w. of A. awamori, respectively, on day 9th and continued receiving these doses daily until day 16th. Rats in the AA group exhibited marked histopathological alterations of the distal colon, with an exaggeration of the DAI. In addition, a remarkable increase in oxidative stress was represented by the elevation of MDA and NO levels with a decline in SOD and GPx activities. In addition, upregulation of TNF-α, IL-6, and IL-1ß mRNA expressions and downregulation of Muc2 and Nrf2 levels were detected. Unambiguously, a remarkable anti-inflammatory effect was noticed either in A. awamori prevented or treated groups expounded by reducing and regulating TNF-α, IL-6, and IL-1ß with improved pathological lesion scoring. The Muc2, Nrf2, and bcl-2 gene levels were upregulated and restored also. In summary, the findings in this work reveal that A. awamori supplementation successfully alleviated the UC induced by AA, which had a better effect when administered before colitis induction.

Valorisation of the invasive alga Rugulopteryx okamurae through the production of monomeric sugars.

Rugulopteryx okamurae is an invasive brown alga causing severe environmental and economic problems on the western Mediterranean coasts. Thus, in addition to the difficulties caused to the fishing and tourism sectors, there is a need to manage its accumulation on the beaches. This work aims to valorise this waste by using it as raw material for producing monosaccharides through a two-stage sequential process. These sugars could be used for different fermentative processes to obtain high-value-added bioproducts. In this work, biological pretreatment of the previously conditioned seaweed with the fungus Aspergillus awamori in solid-state fermentation (SSF), followed by enzymatic hydrolysis with a commercial enzyme cocktail, was performed. The effect of the extension of the biological pretreatment (2, 5, 8 and 12 days) on the subsequent release of total reducing sugars (TRS) in the enzymatic hydrolysis stage was studied. To analyse this effect, experimental data of TRS produced along the hydrolysis were fitted to simple first-order kinetics. Also, the secretion of cellulase and alginate lyase by the fungus, along with the biological pretreatment, was determined. The results suggest that 5 days of biological pretreatment of the macroalgae with A. awamori followed by enzymatic saccharification for 24 h with Cellic CTec2® (112 FP units/g of dry biomass) are the best conditions tested, allowing the production of around 240 g of TRS per kg of dried biomass. The main sugars obtained were glucose (95.8 %) and mannitol (1.5 %), followed by galactose (1 %), arabinose (0.9 %) and fucose (0.5 %). KEY POINTS: • Five-day SSF by A. awamori was the best condition to pretreat R. okamurae. • Five-day SSF was optimal for alginate lyase production (1.63 ±0.011 IU/g biomass). • A maximum yield of 239 mg TRS/g biomass was obtained (with 95.8 % glucose).

Characterization of a new Saccharomyces cerevisiae isolated from banana stems and its mutant with l-leucine accumulation for awamori brewing.

We isolated a new strain of the yeast Saccharomyces cerevisiae, 35a14, from banana stems in Okinawa. This strain did not belong to any industrial yeast groups in a phylogenetic tree and produced high levels of alcohol. Furthermore, awamori, an Okinawa's traditional distilled alcoholic beverage, brewed with an l-leucine overproducing mutant derived from 35a14 showed a high concentration of isoamyl acetate.

Valorization of orange peels exploiting fungal solid-state and lacto-fermentation.

BACKGROUND: Orange peels can serve as a cost-effective raw material for the production of lactic acid. Indeed, given their high concentration of carbohydrates and low content of lignin, they represent an important source of fermentable sugars, recoverable after a hydrolytic step. RESULTS: In the present article, the fermented solid, obtained after 5 days of Aspergillus awamori growth, was used as the only source of enzymes, mainly composed of xylanase (40.6 IU g-1 of dried washed orange peels) and exo-polygalacturonase (16.3 IU g-1 of dried washed orange peels) activities. After the hydrolysis, the highest concentration of reducing sugars (24.4 g L-1 ) was achieved with 20% fermented and 80% non-fermented orange peels. The hydrolysate was fermented with three lactic acid bacteria strains (Lacticaseibacillus casei 2246 and 2240 and Lacticaseibacillus rhamnosus 1019) which demonstrated good growth ability. The yeast extract supplementation increased the lactic acid production rate and yield. Overall, L. casei 2246 produced the highest concentration of lactic acid in mono-culture. CONCLUSION: To the best of our knowledge this is the first study exploiting orange peels as low-cost raw material for the production of lactic acid avoiding the employment of commercial enzymes. The enzymes necessary for the hydrolyses were directly produced during A. awamori fermentation and the reducing sugars obtained were fermented for lactic acid production. Despite this preliminary work carried out to study the feasibility of this approach, the concentrations of reducing sugars and lactic acid produced were encouraging, leaving open the possibility of other studies for the optimization of the strategy proposed here. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Solid state fermentation of fenugreek (Trigonella foenum-graecum): implications on bioactive compounds, mineral content and in vitro bioavailability.

In the present study, solid-state fermentation (SSF) of four fenugreek cultivars viz. HM-57, AFG-2, RMT-1 and RMT-303 were carried out using Aspergillus awamori and its effect on antioxidant properties, phenolic content and bioactive compounds were studied. Macro (Ca, K, and Na) as well as micro (Fe, Zn, and Cu) elements and in vitro bioavailability of the unfermented fenugreek (UFF) and Aspergillus-fermented fenugreek (AFF) samples were assessed with standard methods. On 5th day, total phenolic and condensed tannin contents showed significant (p ≤ 0.05) increase for all cultivars. Further, HPLC analysis confirmed formation of some new bioactive (vanillin, benzoic acid and catechin) compounds. Similarly, extracts from all AFF also showed an increase in the antioxidant potential such as inhibition of DPPH, hydroxyl free radical scavenging, reducing power, and total antioxidant capacity up to 5th day of SSF. Mineral in AFF were found with enhanced values when compared with respective UFF. In vitro bioavailability of Fe, Zn and Ca was also improved during SSF. Results from the present study may be helpful to food industry in developing new health foods and may provide a rational for development of functional ingredient in preparation of novel nutraceuticals.

Beneficial influences of dietary Aspergillus awamori fermented soybean meal on oxidative homoeostasis and inflammatory response in turbot (Scophthalmus maximus L.).

High levels of soybean meal (SBM) in aquafeed leads to detrimental inflammatory response and oxidative stress in fish. In the present study, fermentation with Aspergillus awamori was conducted to explore the potential effects on improving the nutritional quality of soybean meal and the health status of turbot. A 63-day feeding trial (initial weight 8.53 ±â€¯0.11 g) was carried out to evaluate the utilization of fermented soybean meal (FSM) by juvenile turbot. 0% (FM, control), 30% (S30, F30), 45% (S45, F45), and 60% (S60, F60) of fish meal were replaced with SBM or FSM, respectively. As the results showed, fermentation significantly reduced the contents of anti-nutritional factors in SBM, including raffinose (-98.8%), glycinin (-98.5%), ß-conglycinin (-97.4%), trypsin inhibitors (-80%) and stachyose (-80%). A depression of fish growth performance and activities of superoxide dismutase and lysozyme were observed in S45 and S60 groups, while these inferiorities were only observed in F60 group. Meanwhile, fermentation also improved the heights of enterocytes and microvillus significantly in the F45 and F60 groups compared with those in SBM. An induced expression of anti-inflammatory cytokine transforming growth factor-ß and depression of pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1ß in the distal intestine were observed in the F45 and F60 groups. Taken together, this study indicated that fermentation with Aspergillus awamori could improve the replacement level with soybean meal from 30% to 45% in turbot.

Antidiabetic potential of a peptide isolated from an endophytic Aspergillus awamori.

AIM: To exploit the potential of endophytic fungi for pharmaceutically important antidiabetic alpha glycosidase inhibitors. METHODS AND RESULTS: Thirty six endophytic fungi were isolated from Acacia nilotica and screened for the production of alpha amylase and glucosidase inhibitors. Inhibitory activity against both alpha amylase (81%) and alpha glucosidase (80%) was exhibited in an isolate, identified to be Aspergillus awamori. Purification of the inhibitor was carried out on Sephadex LH-20 column and semi prep HPLC. The inhibitor was characterized to be proteinaceous in nature with an approximate molecular mass of 22 kDa. UHPLC amino acid analysis indicated the presence of amino acids serine, threonine, tyrosine and valine in the peptide. The purified inhibitor exhibited mixed type of inhibition against alpha amylase and alpha glucosidase with IC50 values of 3·75 and 5·625 µg ml(-1) respectively. The inhibitor was stable over a wide range of pH and temperature. Optimization of process parameters to increase the yield of the inhibitor was undertaken using one factor at a time approach as well as RSM statistical analysis. The interaction of dextrose and proteose peptone for the test organism was significant with first order effect of pH. Increase of 13% was obtained in the inhibitory activity after optimization of process parameters. Mutagenicity testing by Ames test revealed nonmutagenic nature of the peptide. CONCLUSION: Endophytic A. awamori is capable of producing a peptide with alpha glycosidase inhibitory activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitor obtained in this study possesses dual (alpha glucosidase and alpha amylase) inhibitory activity, low IC50 values, is highly stable under extreme conditions of pH and temperature, and is nonmutagenic in nature. By virtue of its properties it can be commercially produced and exploited for better management of diabetes.

Biodegradation kinetics of thin-stillage treatment by Aspergillus awamori and characterization of recovered chitosan.

An attempt has been made to provide solution for distillery wastewater using fungal pretreatment followed by an anaerobic process to achieve higher organic matter removal, which is a challenge at present with currently adopted technologies. Submerged growth kinetics of distillery wastewater supernatant by Aspergillus awamori was also evaluated. The proposed kinetic models using a logistic equation for fungal growth and the Leudeking-Piret equation for product formation were validated experimentally, and substrate consumption equation was derived using estimated kinetic coefficients. Up to 59.6 % chemical oxygen demand (COD) and 70 % total organic carbon (TOC) removals were observed in 96 h of fungal incubation. Maximum specific growth rate of fungi, coefficient of biomass yield on substrate and growth-associated product formation coefficient were estimated to be 0.07 ± 0.01 h(-1), 0.614 kg biomass/kg utilized COD and 0.215 kg CO2/kg utilized TOC, respectively. The chitosan recovery of 0.072-0.078 kg/kg of dry mycelium was obtained using dilute sulphuric acid extraction, showing high purity and characteristic chitosan properties according to FTIR and XRD analyses. After anaerobic treatment of the fungal pretreated effluent with COD concentration of 7.920 ± 0.120 kg COD/m(3) (organic loading rate of 3.28 kg COD/m(3) day), overall COD reduction of 91.07 % was achieved from distillery wastewater.

Transformation of Litchi Pericarp-Derived Condensed Tannin with Aspergillus awamori.

Condensed tannin is a ubiquitous polyphenol in plants that possesses substantial antioxidant capacity. In this study, we have investigated the polyphenol extraction recovery and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of the extracted polyphenol after litchi pericarp is treated with Aspergillus awamori, Aspergillus sojae or Aspergillus oryzae. We have further explored the activity of A. awamori in the formation of condensed tannin. The treatment of A. awamori appeared to produce the highest antioxidant activity of polyphenol from litchi pericarp. Further studies suggested that the treatment of A. awamori releases the non-extractable condensed tannin from cell walls of litchi pericarp. The total extractable tannin in the litchi pericarp residue after a six-time extraction with 60% ethanol increased from 199.92 ± 14.47-318.38 ± 7.59 µg/g dry weight (DW) after the treatment of A. awamori. The ESI-TOF-MS and HPLC-MS² analyses further revealed that treatment of A. awamori degraded B-type condensed tannin (condensed flavan-3-ol via C4-C8 linkage), but exhibited a limited capacity to degrade the condensed tannin containing A-type linkage subunits (C4-C8 coupled C2-O-C7 linkage). These results suggest that the treatment of A. awamori can significantly improve the production of condensed tannin from litchi pericarp.

Functional Surfaces for Passive Fungal Proliferation Control: Effect of Surface Micro- and Nanotopography, Material, and Wetting Properties.

Fungal proliferation can lead to adverse effects for human health, due to the production of pathogenic and allergenic toxins and also through the creation of fungal biofilms on sensitive surfaces (i.e., medical equipment). On top of that, food spoilage from fungal activity is a major issue, with food losses exceeding 30% annually. In this study, the effect of the surface micro- and nanotopography, material (aluminum, Al, and poly(methyl methacrylate), PMMA), and wettability against Aspergillus awamori is investigated. The fungal activity is monitored using dynamic conditions by immersing the surfaces inside fungal spore-containing suspensions and measuring the fungal biomass growth, while the surfaces with the optimum antifungal properties are also evaluated by placing them near spore suspensions of A. awamori on agar plates. Al- and PMMA-based superhydrophobic surfaces demonstrate a passive-like antifungal profile, and the fungal growth is significantly reduced (1.6-2.2 times lower biomass). On the other hand, superhydrophilic PMMA surfaces enhance fungal proliferation, resulting in a 2.6 times higher fungal total dry weight. In addition, superhydrophobic surfaces of both materials exhibit antifouling and antiadhesive properties, whereas both superhydrophobic surfaces also create an "inhibition" zone against the growth of A. awamori when tested on agar plates.

Use of Awamori-pressed Lees and Tofu Lees as Feed Ingredients for Growing Male Goats.

Awamori is produced by fermenting steamed indica rice. Awamori-pressed lees is a by-product of the Awamori production process. Tofu lees is a by-product of the Tofu production process. Research was conducted to test if dried Awamori-pressed lees and Tofu lees can be used as a mixed feed ingredient for raising male goats. Eighteen male kids were divided into three groups of six animals (control feed group (CFG), Awamori-pressed lees mixed feed group (AMFG), Tofu lees mixed feed group (TMFG)). The CFG used feed containing 20% soybean meal as the main protein source, while the AMFG and TMFG used feed mixed with 20% dried Awamori-pressed lees or dried Tofu lees. The groups were fed mixed feed (volume to provide 100 g/d increase in body weight) and alfalfa hay cubes (2.0 kg/d) twice a day (10:00, 16:00). Klein grass hay and water was given ad libitum. Hay intake was measured at 10:00 and 16:00. Body weight and size measurements were taken once a month. At the end of the experiment, a blood sample was drawn from the jugular vein of each animal and the carcass characteristics, the physical and chemical characteristics of loin were analyzed. DCP and TDN intakes in AMFG and TMFG showed no significant difference to the CFG. Cumulative measurements of growth in body weight and size over the 10 mo period in the AMFG and TMFG were similar to the CFG. Blood parameter values were similar to those in normal goats. Dressing carcass weight and percentages, and total weight of meat in the AMFG were similar to that in the CFG, but smaller in the TMFG. The compressed meat juice ratio was higher in both the TMFG and AMFG than the CFG. While the fat in corn, Awamori-pressed lees, and Tofu lees contains more than 50% linoleic acid, the loin fat in both the AMFG and TMFG was very low in linoleic acid due to the increase in the content of oleic acid, stearic acid, and palmitic acid. This indicates that feeding on AMF and TMF does not inhibit hydrogenation by ruminal microorganisms. As in the CFG, the total essential and non-essential amino acids in the loin of the AMFG and TMFG were well balanced. Compared to the CFG, the AMFG and TMFG were high in taurine and carnosine. The results indicate dried Awamori-pressed lees and Tofu lees can be used as a feed ingredient for raising male goats.

Single substitution in α-helix of active center enhanced thermostability of Aspergillus awamori exo-inulinase.

Exo-inulinases are applied in inulin hydrolysis and production of feed additives and need to be stable at temperatures of 60-95 °C. Aspergillus awamori exo-inulinase Inu1 is considerably thermostable, with a Tm of 73.2 °C. However, the thermostability of the enzyme should be improved. A single substitution G338A in α-helix in the active center of the enzyme provided a 3.5 °C improvement in Tm. The time of half-life at 70 °C and 80 °C was increased in 5.7- and 2.7-times, respectively, compared to wild-type. Molecular dynamics simulations demonstrated that the substitution G338A caused a decrease in RMSF not only for the α-helix 337-YAANI-341, but also for the catalytically active residues D41 and E241 and the amino acid residues forming the cleft of the active center. Calculations with Constraint Network Analysis for the variant G338A showed the increase in the stability of intramolecular clusters.

Electro-stimulation of tofu wastewater for the production of single cell protein from various microorganisms.

Tofu wastewater can be utilized as a substrate for microorganisms that produce single-cell proteins (SCPs). Because different microorganisms have different cellular components, the composition of SCPs varies. Electro-stimulation has the potential to speed up fermentation and increase product yield. The goal of this study was to find the best way to produce SCPs from Aspergillus awamori, Rhizopus oryzae, and Saccharomyces cerevisiae in the tofu wastewater substrate using electro-stimulation. The experimental method was used in the study, the data were analyzed using independent t-test statistical analysis, and the best treatment was identified using the effective index method. This treatment consisted of producing SCP with electro-stimulation of -1.5 V and without electro-stimulation for 72 h for the yeast and 96 h for the mold at 25 °C in tofu wastewater that had already been conditioned to a pH of 5. The parameters measured included measurement of population of microorganism, change in pH, dry biomass weight, carbohydrate content, and protein content. Electro-stimulation reduced the optimum fermentation time of A. awamori SCP from 56 to 32 h, resulting in 0.0406 g/50 mL of dry biomass, 30.09% carbohydrate content, and 6.86% protein content. Meanwhile, the optimal fermentation time on R. oryzae and S. cerevisiae were not accelerated by electro-stimulation. The best treatment was A. awamori without electro-stimulation, which produced 0.0931 g/50 mL of dry biomass, 20.29% carbohydrate, and 7.55% protein.

Use of Awamori-pressed Lees and Tofu Lees as Feed Ingredients for Growing Female Goats.

Okinawan Awamori is produced by fermenting steamed indica rice with black mold, yeast, and water. Awamori-pressed lees is a by-product of the Awamori production process. Tofu lees is a by-product of the Tofu production process. This research consisted of two experiments conducted to elucidate whether or not dried Awamori-pressed lees and Tofu lees can be used as a mixed feed ingredient for raising female goats. In experiment 1, digestion trials were conducted to ascertain the nutritive values of dried Awamori-pressed lees and dried Tofu lees for goats. The digestible crude protein (DCP) and total digestible nutrients (TDN) contents of dried Awamori-pressed lees and Tofu lees were 22.5%, 22.5% (DCP), and 87.2%, 94.4% (TDN) respectively. In experiment 2, 18 female goats (Japanese Saanen×Nubian, three months old, body weight 15.4±0.53 kg) were divided into three groups of six animals (control feed group (CFG), Awamori-pressed lees mixed feed group (AMFG), Tofu lees mixed feed group (TMFG)). The CFG control used feed containing 20% soybean meal as the main protein source, while the AMFG and TMFG treatments used feed mixed with 20% dried Awamori-pressed lees or dried Tofu lees. The groups were fed mixed feed (volume to provide 100 g/d increase in body weight) twice a day (10:00, 16:00). The klein grass hay and water was given ad libitum. The hay intake was measured at 08:00 and 16:00. Body weight and size measurements were taken once a month. At the end of the experiment, a blood sample was drawn from the jugular vein of each animal. The DCP and TDN intakes in AMFG and TMFG showed no significant difference to the CFG. Cumulative measurements of growth in body weight, withers height, chest depth, chest girth, and hip width over the 10 mo period in the AMFG and TMFG were similar to the CFG. By contrast, cumulative growth in body length and hip height in the AMFG and TMFG tended to be larger than the CFG. Cumulative growth in chest width in the AMFG was significantly larger (p<0.05) than the CFG. Blood parameter values were similar to those in normal goats reported by other scientists. The coats of the AMFG and TMFG animals looked shinier than the animals in the CFG. The results demonstrate that dried Awamori-pressed lees and Tofu lees can be used as a feed ingredient for the raising female goats.

Identification of 2-furanmethanethiol contributing to roast aroma in honkaku shochu and awamori.

Honkaku shochu and awamori are traditional Japanese spirits. 2-Furanmethanethiol (2FM), a volatile thiol, was identified as a roast aroma compound in honkaku shochu and awamori. The detection threshold of 2FM in 25% (v/v) ethanol water solutions was determined as 1.6 ng/L. The odor activity values, calculated using the detection threshold suggested that 2FM affects the quality of honkaku shochu and awamori. The odor activity values of 2FM were higher in barley shochu distilled at atmospheric pressure than in sweet potato shochu, rice shochu and awamori; therefore, 2FM is considered to contribute to the characteristics of barley shochu.

Aspergillus awamori attenuates ochratoxin A-induced renal and cardiac injuries in rabbits by activating the Nrf2/HO-1 signaling pathway and downregulating IL1ß, TNFα, and iNOS gene expressions.

Ochratoxin A (OTA) is one of the most dangerous and that pollute agricultural products, inducing a variety of toxic effects in humans and animals. The current study explored the protective effect of different concentrations of Aspergillus awamori (A. awamori) against OTA (0.3 mg/kg diet) induced renal and cardiac damage by exploring its mechanism of action in 60 New Zealand white male rabbits. Dietary supplementation of A. awamori at the selected doses of 50, 100, and 150 mg/kg diet, respectively, for 2 months significantly improved the rabbit's growth performance; modulated the suppressed immune response and restored the altered hematological parameters; reduced the elevated levels of renal injury biomarkers such as urea, creatinine, and alkaline phosphatase; and increased serum total proteins concentrations. Moreover, it also declined enzymatic activities of cardiac injury biomarkers, including AST, LDH, and CK-MB. A. awamori alleviated OTA-induced degenerative and necrotic changes in the kidney and heart of rabbits. Interestingly, A. awamori upregulated Nrf2/OH-1 signaling pathway. Therefore enhanced TAC, CAT, and SOD enzyme activities and reduced OTA-induced oxidative and nitrosative stress by declining iNOS gene expression and consequently lowered MDA and NO levels. In addition to attenuating renal and cardiac inflammation via reducing IL-1ß, TNF-α gene expressions in a dose-dependent response. In conclusion,this is the first report to pinpoint that dietary incorporation of A. awamori counteracted OTA-induced renal and cardiac damage by potentiating the rabbit's antioxidant defense system through its potent antioxidant, free radical scavenging, and anti-inflammatory properties in a dose-dependent response. Based on our observations, A. awamori could be utilized as a natural protective agent against ochratoxicosis in rabbits.

Antifungal Activity of Thymol against Aspergillus awamori and Botrytis aclada Isolated from Stored Onion Bulbs.

The antifungal activity of thymol against Aspergillus awamori F23 and Botrytis aclada F15 in onions was examined through direct treatment with amended media and gaseous treatment with I-plates (plastic plates containing central partitions). The protective and curative control efficacy of thymol was examined 24 h before and after the inoculation of onion bulbs with the fungal isolates. Mycelial growth, sporulation, and spore germination of the isolates were inhibited on potato dextrose agar amended with various concentrations of thymol or acetic acid (positive control). Overall, thymol produced a stronger inhibitory effect on the mycelial growth and development of the isolates than acetic acid. Following gaseous treatment in I-plates, mycelial growth, sporulation, and spore germination of the isolates were inhibited at higher concentrations of thymol or acetic acid; however, acetic acid showed a little effect on the sporulation and spore germination of the isolates. Following the treatment of onion bulbs with 1000 mg L-1 of thymol 24 h before and after fungal inoculation, lesion diameter was greatly reduced compared with that following treatment with 0.5% ethanol (solvent control). Onion bulbs sprayed with thymol 24 h before fungal inoculation generally showed reduced lesion diameters by isolate F23 but not in isolate F15 compared with those sprayed 24 h after fungal inoculation. Collectively, thymol effectively inhibited the growth and development of A. awamori and B. aclada on amended media and in I-plates. In addition, spraying or fumigation of thymol is more desirable for effectively controlling these postharvest fungal pathogens during long-term storage conditions.

Study of protease activity from Aspergillus awamori INCQS2B.361U2/1 extracellular fraction and modification of culture medium composition to isolate a novel aspartic protease.

Aspergillus awamori was cultivated in a modified Breccia medium, and the extracellular fraction was obtained, which presented 260 ± 15 µg of protein/mg and specific protease activity of 3.87 ± 0.52 mM.min-1.mg of protein-1 using Nα-p-tosyl-L-arginine methyl ester hydrochloride (L-TAME) as substrate. This fraction showed major proteins about 104 and 44 kDa and maximal protease activity at pH 5.5, 6.5, and 9.0, suggesting that A. awamori secretes acidic, neutral, and alkaline proteases with expressive thermal stability, however, aspartic protease was the most important activity. When yeast extract was supplemented to a modified Breccia medium, A. awamori protein secretion and protease activity were maximal and the affinity chromatography on pepstatin-agarose was employed to isolate the aspartic protease activity, which was called ASPA, with approximately 75 kDa. ASPA maximal activity was obtained at pH 4.5 and 6.5, and 50 °C. Pepstatin inhibited about 80% of ASPA activity, with IC50 and Ki values of 0.154 and 0.072 µM, respectively. ASPA cleaved protein and peptides substrates with the highest activity against gelatin (95 U/mg) and good peptidase activity with KM 0.0589 mM and Vmax 1.909 mM.min-1.mg protein-1, using L-TAME as substrate. A. awamori extracellular fraction is a source of proteases with important activity, and the supplementation of modified Breccia medium increased the aspartic protease production. This enzyme presented different biochemical characteristics from the previously reported A. awamori aspartic proteases. Therefore, ASPA is an excellent candidate for biotechnological application due to its important activity and thermostability.