Relationship Between Extracellular Cellulase,Pectinase and Xylanase Activity of Isolated Aspergillus oryzae Strains Grown on Koji and the Umami-Tasting Amino Acid Content of Soy Sauce

We compared the koji-based extracellular carboxymethylcellulases (CM-cellulase), pectinases and xylanases of four Aspergillus oryzae isolates from soy sauce koji, A1, PRB-1 (A2), A3 and A4, with Aspergillus oryzae AS3.951 (A5) via enzyme assays and zymography and determined their correlation to the content of umami-tasting amino acids in high-salt diluted-solid state growth stage leading to soy sauce. In A2 koji, xylanase had the highest activity (28.75 U/g dry weight). Zymogram analysis revealed two different bands of CM-cellulase activity (termed Cel-1 and Cel-2) and one band of pectinase activity (Pec-1) in all koji samples. The intensity of these bands was comparable among all five strains. Interestingly, the same analysis revealed six different xylanase activity bands (termed Xyl-1 to Xyl-6) in all koji samples. After 60 days of cultivation, the free amino acid concentration was highest in soy sauce generated by A4 at 6709.41 mg/100 ml and contained 1690.82 mg/100 ml umami-tasting amino acids (including glutamic acid and aspartic acid), followed by soy sauce generated by A2. Pearson’s coefficient analysis indicated that Xyl-1 and Xyl-2 activities were positively associated with the umami-tasting amino acid content of soy sauce. In conclusion, these data suggested that the xylanases Xyl-1 and Xyl-2 in koji could be important contributors to the umami taste formation of soy sauce.     

Properties of cellulose-degrading enzymes from Aspergillus oryzae and their contribution to material utilization and alcohol yield in sake mash fermentation

Four cellulose-degrading enzymes were identified in a solid-state culture of Aspergillus oryzae. The three major enzymes were purified and named Cel-1, Cel-2, and Cel-3, respectively. The molecular weights were determined to be 62, 120, and 34 kDa, respectively. The optimum temperature of Cel-3 activity was higher than that of the other enzymes. An acidic pH was found to be more suitable for Cel-1 activity than for the other enzymes, and Cel-3 was more stable under acidic conditions than the other two. These properties and the results of a protein homology search for N-terminal amino acid sequences suggest that Cel-1 and Cel-3 correspond to the previously isolated endo-1,4-beta-glucanase CelB and CelA, respectively. The analysis of substrate specificity suggested that Cel-2 is likely to be beta-glucosidase. The effect of Cel-1, Cel-2, and Cel-3 on the sake mash fermentation was determined and it was found that Cel-2 markedly improved material utilization and alcohol yield in sake mash fermentation.     

Identification and toxigenic potential of the industrially important fungi, Aspergillus oryzae and Aspergillus sojae

Mold strains belonging to the species Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu, and as protein production hosts in modern industrial processes. A. oryzae and A. sojae are relatives of the wild molds Aspergillus flavus and Aspergillus parasiticus. All four species are classified to the A. flavus group. Strains of the A. flavus group are characterized by a high degree of morphological similarity. Koji mold species are generally perceived of as being nontoxigenic, whereas wild molds are associated with the carcinogenic aflatoxins. Thus, reliable identification of individual strains is very important for application purposes. This review considers the pheno- and genotypic markers used in the classification of A. flavus group strains and specifically in the identification of A. oryzae and A. sojae strains. Separation of A. oryzae and A. sojae from A. flavus and A. parasiticus, respectively, is inconsistent, and both morphologic and molecular evidence support conspecificity. The high degree of identity is reflected by the divergent identification of reference cultures maintained in culture collections. As close relatives of aflatoxin-producing wild molds, koji molds possess an aflatoxin gene homolog cluster. Some strains identified as A. oryzae and A. sojae have been implicated in aflatoxin production. Identification of a strain as A. oryzae or A. sojae is no guarantee of its inability to produce aflatoxins or other toxic metabolites. Toxigenic potential must be determined specifically for individual strains. The species taxa, A. oryzae and A. sojae, are currently conserved by societal issues.     

Changes in selected physical property and enzyme activity of rice and barley koji during fermentation and storage

Koji are solid-state fermentation products made by inoculating steamed grains with the spores of fungi, particularly Aspergillus spp. This research was undertaken to identify the fermentation and storage conditions optimal for the production and maintenance of selected hydrolytic enzymes, such as α-amlyase and protease, in koji. Steamed rice and barley were inoculated with 2 × 10 (11) Aspergillus oryzae spores per kilogram of grains and fermented for 118 h in a growth chamber at 28 to 32 °C with controlled relative humidities. Samples were drawn periodically during fermentation and storage at -20, 4, or 32 °C, and α-amylase and protease activity, mold counts, a(w), moisture contents, and pH of collected samples were determined. It was observed that the a(w), moisture contents, and pH of the koji were influenced by the duration of fermentation and temperature of storage. The α-amylase activity of both koji increased as the populations of A. oryzae increased during the exponential growth phase. The enzyme activity of barley koji was significantly higher than that of rice koji, reaching a peak activity of 211.87 or 116.57 U at 46 and 58 h, respectively, into the fermentation process. The enzyme activity in both products started to decrease once the mold culture entered the stationary growth phase. The protease activities of both koji were low and remained relatively stable during fermentation and storage. These results suggest that rice and barley koji can be used as sources of α-amylase and desired enzyme activity can be achieved by controlling the fermentation and storage conditions. PRACTICAL APPLICATION: Amylases and proteases are 2 important hydrolytic enzymes. In the food industry, these enzymes are used to break down starches and proteins while reducing the viscosity of foods. Although amylases and proteases are found in plants and animals, commercial enzymes are often produced using bacteria or molds through solid state fermentation, which is designed to use natural microbial process to produce enzymes in a controlled environment. A properly produced and maintained koji with a high hydrolytic enzyme activity can serve as an important source of the enzymes for the food industry.     

PRODUCTION AND CHARACTERISTICS OF RED RICE SAKE

Red rice sake was efficiently produced from unpolished red rice without cooking by use of rice koji prepared with shochu koji mold, Aspergillus kawachii, as saccharifying agent. A comparative study was made of red rice sake made with rice koji and red rice wine made with a preparation of glucoamylase known as Sumizyme. Red rice sake contained about 13% ethanol and its acidity was 8.6. Large amounts of volatile compounds, such as isobutyl alcohol and ethyl acetate, were found in the red rice sake. The red color of the red rice sake was more intense than that of the red rice wine. The quality of red rice sake was better than that of red rice wine, as assessed by gas chromatography, spectrophotometric analysis and organoleptic testing. In accordance with the traditional method for sake brewing, red rice, rice koji and tap water were added to the initial mash. The red color of the red rice sake made with these additions was more intense than that of red rice sake made without these additions. The rice wine was somewhat improved by these additions, as assessed by organoleptic testing. The body of the resultant rice wine was fortified with dextrin and its mild characteristics were derived from sugar and dextrin.     

Secretome of Aspergillus oryzae in Shaoxing rice wine koji

Shaoxing rice wine is the most famous and representative Chinese rice wine. Aspergillus oryzae SU16 is used in the manufacture of koji, the Shaoxing rice wine starter culture. In the current study, a comprehensive analysis of the secretome profile of A. oryzae SU16 in Shaoxing rice wine koji was performed for the first time. The proteomic analysis for the identification of the secretory proteins was done using two-dimensional electrophoresis combined with matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry based on the annotated A. oryzae genome sequence. A total of 41 unique proteins were identified from the secretome. These proteins included 17 extracellular proteins following the classical secretory pathway, and 10 extracellular proteins putatively secreted by the non-classical secretory pathway. The present secretome profile greatly differed from previous reports on A. oryzae growing in other solid-state nutrient sources. Several new secretory or putative secretory proteins were also found. These proteomic data will significantly aid the advancement of research on the secretome of A. oryzae, especially in solid-state cultures, and in elucidating the production process mechanism of Shaoxing rice wine koji. The findings may promote the technological development and innovation of the Shaoxing rice wine industry.     

Effect of sake lees on cheese components in cheese ripened by Aspergillus oryzae and lactic acid bacteria

More than 2,000 varieties of cheese currently exist in the world, and cheese manufacture continues to flourish. To develop the cheese ripening process, additional ingredients are used during cheese production. In this study, the effect of sake lees as an additional ingredient on the fermentation of cheese using Aspergillus oryzae (koji mold), known as koji cheese, was investigated. Aspergillus oryzae is used in the fermentation of Japanese traditional foods, such as sake and soy sauce, given its strong enzymatic activities, as well as in cheese production (i.e., koji cheese). Sake lees, a by-product of the fermentation of rice with A. oryzae and yeasts in the sake brewing process, contains various metabolites, such as amino acids. Here, supplementation with sake lees enhanced the activities of lactic acid bacteria and affected the color of the cheese. Metabolome analysis revealed that sake lees altered the balance of carbohydrates and fatty acids in the cheese. Remarkably, supplementation with sake lees enhanced the production of umami-enhancing γ-glutamyl (kokumi-active) peptides. This study suggests that a new type of cheese can be produced using A. oryzae and sake lees, and information on the synergistic effects of A. oryzae and sake lees will aid the development of cheese production.     

Analysis of Volatile Compounds in Shochu Koji,Sake Koji,and Steamed Rice by Gas Chromatography‐Mass Spectrometry

The volatile compounds in three types of koji (yellow, white, and black) and steamed rice were investigated using gas chromatography‐mass spectrometry. The objective of this study was to characterize each koji type based on its profile of volatile compounds. From the steamed rice and rice koji samples examined, a total of 29 volatile compounds were identified. Butanal, hexanal, dimethyl trisulfide and cyclohexyl isothiocyanate were detected only in steamed rice. The volatile profile of yellow koji (sake koji) was distinct from those of white and black kojis (shochu koji). On the basis of relative peak areas, alcohols and aldehydes were identified as being more abundant in yellow koji. White and black kojis had similar volatile profiles, and had a greater abundance of furans than yellow koji. We assume that the different suites of volatile compounds identified in shochu and sake kojis impart the respective characteristic flavours to shochu and sake.     

酿酒ADY在黄酒生产中的应用

黄酒活性干酵母实现了酒母商品化，是酒母培养工艺的重大创新，对促进黄酒工业现代化具有重要意义。论述了黄酒活性干酵母在机制麦曲黄酒、乌衣红曲酒和红曲酒、传统黄酒、酒母扩大培养、夏季籼米黄酒生产中的应用，以及AADY在米曲生产中的应用等。     

六粮风味米酒工艺研究

以玉米、大米、小米、黑米、糯米、糯玉米6种米为原料,以米根霉和酿酒活性干酵母为糖化发酵剂,通过单因素试验对6种淀粉质原料米酒酿造用麸曲和酒曲的种类、比例及接种量、发酵温度、发酵时间等进行研究,再采用正交试验优选出六粮风味米酒发酵的最佳工艺参数。结果表明:麸曲与酒曲最佳质量比为1∶1,加曲量为0.6%,30℃条件下发酵48 h;玉米∶大米∶小米∶黑米∶糯米∶糯玉米的质量比为1∶1∶1∶2∶2∶2。此时的米酒质量最佳,酒质地均匀、酒醪清澈、醇香浓郁,柔和爽口,具有独特的甜米酒风味。     

菌种耦合发酵米渣生酱油的品质和抗氧化活性

以高盐稀态法酿造米渣生酱油,探究菌种耦合对其品质和抗氧化活性的影响。4组不同菌种耦合发酵的米渣生酱油品质测定结果表明其各项质量指标均达到GB 18186—2000《酿造酱油》中一级酱油的标准,其中S4生酱油(米曲霉耦合黑曲霉和鲁氏酵母发酵)游离氨基酸总量达到41.81 g/L,4种米渣生酱油的总氨基酸、必需氨基酸、呈味氨基酸和抗氧化氨基酸的质量浓度均高于同时发酵的大豆生酱油S5生酱油(米曲霉发酵)。S3生酱油(米曲霉耦合鲁氏酵母发酵)和S4生酱油的抗氧化活性普遍高于S1生酱油(米曲霉发酵)和S2生酱油(米曲霉耦合黑曲霉发酵),各结果均表明S3的抗氧化活性最强,清除羟自由基的能力达到等质量浓度VC的最高175.42倍,对1,1-二苯基-2-三硝基苯肼自由基清除能力达到VC的2.16倍。抗氧化活性受发酵温度影响明显,分别在35℃和50℃发酵时出现峰值。多菌种耦合发酵对米渣生酱油品质和抗氧化活性均有明显改善,其中鲁氏酵母发挥了明显作用。     

RICE WINE BREWING WITH SPROUTING RICE,SPROUTING RICE INFECTED WITH ASPERGILLUS ORYZAE AND RICE KOJI

Fermentation tests were performed with sprouting rice, sprouting rice infected with koji mold, i.e. Aspergillus oryzae and rice koji, as saccharifying agent. Characteristics of the various rice wines were compared by gas chromatography and organoleptic testing. The rice wine made with sprouting rice was colorless and had light and refined characteristics in terms of both aroma and taste. The rice wine made with sprouting rice infected by Aspergillus oryzae was rich in isoamyl alcohol, isoamyl acetate and acetaldehyde; it was faintly yellow in color and had rich and mild characteristics. This rice wine had something in common with conventional Japanese sake as compared with the sprouting rice wine, and it might be a transitional intermediate between ancient sprouting rice wine and conventional Japanese sake. In accordance with the traditional method for sake brewing, starchy materials, tap water and sprouting rice as the saccharifying agent were added to the initital mash as first and second additions. The rice wine was somewhat improved by these additions, as assessed by organoleptic testing. The body of the resultant rice wine was fortified with dextrin and its acidic taste was masked by sugar and dextrin.     

响应面优化香菇柄发酵酱糕的制曲工艺

为了制备香菇柄发酵酱糕,以糯米、黄豆为原料,选用米曲霉（Aspergillus oryzae）As3.042进行制曲。以蛋白酶活力、淀粉酶活力、纤维素酶活力为评价指标,对原料比（糯米∶黄豆）、接种量、培养时间、培养温度进行单因素试验优化,并在单因素试验的基础上对发酵酱糕的制曲工艺进行响应面优化。结果表明,香菇柄发酵酱糕的制曲最优条件为糯米与黄豆质量比2∶3、米曲霉接种量0.8%、培养时间48 h、培养温度30 ℃,此条件下优化后的成曲蛋白酶活力为2 625.48 U/g。     

板栗大米清酒酒曲选择及氨基酸含量测定

为了确定板栗作为酿酒原料的可行性,对板栗淀粉糖化用曲进行了筛选,同时分析板栗大米清酒中氨基酸的种类及含量,为 清酒的营养与风味的改良提供基础。 通过对4种米曲霉（Aspergillus oryzae）酒曲的感官评价及酶活测定,确定米曲霉2011更适合作为 板栗清酒的糖化曲种,其α-淀粉酶活力、糖化酶活力及酸性蛋白酶活力分别为1 306.63 U/g、324.56 U/g、1 036.33 U/g。 板栗大米清酒 及纯大米清酒中氨基酸含量分析结果表明,板栗大米清酒含有17种氨基酸（含8种必需氨基酸）,且含量均高于大米清酒中的氨基酸 含量,其中天冬氨酸、苏氨酸、谷氨酸、丙氨酸、缬氨酸、异亮氨酸、亮氨酸、酪氨酸、苯丙氨酸含量是大米清酒中的2倍以上。 添加板栗 不仅改良了清酒的营养价值,还改善了清酒的风味,因此板栗适合作为酿酒原料。     

Identification of 2,4,6-trichloroanisole (TCA) causing a musty/muddy off-flavor in sake and its production in rice koji and moromi mash

2,4,6-Trichloroanisole (TCA), which has been identified as the main component responsible for the cork taint in wine, was detected in sake samples having a musty/muddy off-flavor by stir bar sorptive extraction (SBSE). We confirmed that TCA is one of the components causing this off-flavor in sake, as in other alcoholic beverages, from a sensory analysis showing the correlation between TCA concentration and the intensity of the musty/muddy off-flavor. We investigated the route of TCA production in the rice koji preparation process and in the moromi mash process for sake brewing. We found that TCA is produced mainly by the biomethylation of 2,4,6-trichlorophenol (TCP) by rice koji in brewing and that TCP originates from the wooden tools used in preparing rice koji.     

泰国甜酒曲中优势真菌的分离鉴定初报

对泰国优质甜酒曲中4株优势真菌（霉菌菌株编号为R1、R2，酵母菌株编号为L1、L2）进行了分离、纯化培养，旨在为开发更有价值的甜酒菌株提供实验依据。鉴定结果表明：霉菌菌株R1为米根霉种（Rhizopus Oryzae）的真菌，R2为华根霉种（Rhizopus chinensis）的真菌；酵母菌株L1、L2为假丝酵母属（Candida）的酵母菌。     

两种生产用米曲霉的形状及蛋白酶性质研究

研究对比了两株米曲霉的形态特征以及成曲的蛋白酶酶学性质。结果显示,两株米曲霉的形态差异明显:米曲霉1号菌落较大、发芽和产孢子较快,种曲及成曲颜色偏黄,菌丝较短,孢子较多;而米曲霉2号菌落较小,孢子发芽时间及产孢子时间比1号晚,成曲孢子较1号少,但菌丝长且粗壮,种曲颜色偏绿,成曲颜色偏白,米曲霉2号的产酶能力强。蛋白酶性质的研究结果显示,两株米曲霉成曲的蛋白酶性质有差异,1号和2号的最适反应温度分别为45℃和50℃,最适反应pH分别为6.0和9.0。此外,两株米曲霉的成曲蛋白酶的耐盐性相当,随着盐浓度的增大,酶活力均逐渐降低,在20%氯化钠条件下,酶活力均降至无盐条件下的20%~30%。     

A second pectin lyase gene (pel2) from Aspergillus oryzae KBN616: its sequence analysis and overexpression, and characterization of the gene products

A second pectin lyase gene, designated pel2, was isolated from a shoyu koji mold Aspergillus oryzae KBN616 and characterized. The structural gene comprised 1306 bp with three introns. The ORF encoded 375 amino acids with a signal peptide of 19 amino acids. The deduced amino acid sequence showed high similarity to those of A. oryzae Pel1, Aspergillus niger pectin lyases and Glomerella cingulata Pn1A. The pel2 gene was overexpressed under the control of the promoter of the A. oryzae TEF1 gene for purification and enzymatic characterization of its gene product. The gene product exhibited two molecular masses of 48 and 44 kDa due to different degrees of glycosylation. Both proteins had the same pH optimum of 6.0 and temperature optimum of 50 degrees C.     

米发糕胃肠水解物抗氧化性及肽段差异性分析

以籼米粉为原料,三种发酵剂(酵母菌ZSM-001;酵母菌ZSM-001与米根霉ZSM-003;安琪酵母与酒曲发酵剂)制作米发糕,通过凝胶色谱测定其水提物和胃肠水解物分子质量分布,基于液相色谱-四级杆飞行时间质谱(TOF LC/MS/MS)研究肽的差异性,并进行胃肠水解物的抗氧化活性评价。结果表明,米发糕经胃肠水解后,其抗氧化活性提升,分子质量集中在3 ku以下,小分子组分(分子质量3 ku)的Fe~(2+)螯合能力和还原能力较其他组分高。发酵剂组合对米发糕胃肠水解物的肽段数量、疏水性和抗氧化性有显著影响,以安琪酵母与酒曲复合发酵制备米发糕的胃肠水解物中分子质量小于3 ku组分的肽段数最多,疏水性较强,且Fe~(2+)螯合能力最大,还原能力较好。     

甜酒曲中霉菌18SrDNA克隆及其序列分析

以甜酒曲霉菌为研究靶点,提取霉菌基因组DNA,利用PCR 技术扩增该霉菌18SrDNA 部分基因序列,构建pKRX-T 筛选载体,转化E.coil HB101。经测序和相似性分析,结果发现该霉菌菌株与米根霉(Rhizopus oryzae)有99% 同源性,推测该菌可能为米根霉的突变株。