Effects of a mixed koji culture of Aspergillus oryzae HG‐26 and Aspergillus niger HG‐35 on the levels of enzymes,antioxidants and phenolic compounds in soy sauce during the fermentation process

Soy sauce contains a number of bioactive components, which have been shown to possess strong antioxidative properties. The objective of this study was to compare the enzyme activities, antioxidant activity and phenolic compounds content of soy sauces prepared from a mixed koji (SSAON, inoculated with Aspergillus oryzae HG‐26 and Aspergillus niger HG‐35), with those of soy sauces made from a koji culture containing only Aspergillus oryzae HG‐26 koji (SSAO). The total phenolic content (TPC), total flavonoid content (TFC), content of three isoflavone aglycones (daizein, glycitein and genistein) and the antioxidant activity of the SSAON were found to be higher than those of the SSAO during moromi fermentation. In addition, they showed strong positive correlations with the antioxidant potential of the soy sauce. This study has demonstrated the potential of a mixed‐culture koji, based upon A. orzyae and A. niger, for the production of soy sauce with an increased level of bioactive components.     

Effect of mixed kojis on physiochemical and sensory properties of rapid‐fermented fish sauce made with freshwater fish by‐products

The objective of this study was to investigate the effect of mixed kojis on essential indices of fish sauce. Fish sauce inoculated with mixed kojis (FSB, Aspergillus oryzae koji:Aspergillus niger koji = 3:1) and A. oryzae koji (FSA, control) was prepared. At the end of fermentation, the content of amino nitrogen, total soluble nitrogen, Free amino acid (FAA) and glutamic acid in FSB increased by 7.50%, 7.84%, 9.80% and 28.43% compared to FSA, due to higher acid protease activity in mixed kojis. Moreover, flavour groups including phenols, ketones and nitrogen‐containing compounds in FSB were also improved. According to the results of sensory evaluation, FSB showed higher intensity of umami and caramel attributes, lower intensity of ammonia and sour attributes. All the results suggested that the usage of mixed kojis to produce fish sauce was an effective method that can accelerate the fermentation process and improve the flavour of fish sauce made with freshwater fish by‐products.     

Effect of koji fermentation on generation of volatile compounds in soy sauce production

The generation of aroma compounds and the changes in sensory characteristics through the soy sauce koji fermentation were investigated to evaluate the crucial impact of the koji fermentation on soy sauce aroma. The sensory analysis showed a marked increase in ‘musty’ and ‘soy sauce‐like’ odours, whereas the beany attribute decreased significantly during koji fermentation. Aldehydes and alcohols were the major volatile classes present in the koji samples. Gas chromatography–olfactometry was used to identify the aroma‐active compounds in harvested koji, and the results showed that 1‐octen‐3‐ol, 3‐octanol, (E)‐2‐octenal, di‐epi‐α‐cedrene, benzeneacetaldehyde and 3‐(methylthio)‐propanal exhibited the greatest aroma intensities in harvest koji. Moreover, relative contents of (E)‐2‐octenal, benzeneacetaldehyde, 3‐(methylthio)‐propanal and 2‐phenylpropenal were found to be positively related to ‘musty’ and ‘soy sauce‐like’ odours. Results from principal component analysis showed that the growth and sporulation of Aspergillus oryzae had the important impacts on the volatile evolution.     

先固后稀添加酵母、乳酸菌是提高酱油风味的捷径

本文阐述了先固后稀醇造工艺中添加酵母，乳酸菌，采用淋浇后熟发酵原池浸出法可以提高酱油风味；同时采用双菌种制曲提高酱油谷氨酸含量。严格低盐固态发酵工艺规程，保证前期发酵的酱醪质量。该工艺适用于现有低盐固态发酵设备的企业，为国内多数酿造厂生产优质酱油提供了一条合理化建议。     

Effects of rice koji inoculated with Aspergillus luchuensis on the biochemical and sensory properties of a sailfin sandfish (Arctoscopus japonicus) fish sauce

The effects of rice koji inoculated with Aspergillus luchuensis on the biochemical and sensory properties of fish sauce produced from sailfin sandfish (Arctoscopus japonicus) with different salt concentrations (10% and 20%) were investigated over 10 months. Fish sauces prepared from sailfin sandfish with rice koji and a low salt concentration had higher total nitrogen, amino nitrogen and total free amino acid than did those without rice koji or with a high salt concentration. Sensory evaluation of fish sauces prepared with rice koji and a low salt concentration found better taste, flavour and overall acceptance than those prepared without rice koji during fermentation (P < 0.05). The use of rice koji increased the total nitrogen, amino nitrogen and free amino acid and also enhanced the sensory qualities during fermentation. These results suggest that the use of rice koji can result in a low‐salt fish sauce with better flavour.     

Monitoring of Yeast Communities and Volatile Flavor Changes During Traditional Korean Soy Sauce Fermentation

Flavor development in soy sauce is significantly related to the diversity of yeast species. Due to its unique fermentation with meju, the process of making Korean soy sauce gives rise to a specific yeast community and, therefore, flavor profile; however, no detailed analysis of the identifying these structure has been performed. Changes in yeast community structure during Korean soy sauce fermentation were examined using both culture‐dependent and culture‐independent methods with simultaneous analysis of the changes in volatile compounds by GC‐MS analysis. During fermentation, Candida, Pichia, and Rhodotorula sp. were the dominant species, whereas Debaryomyces, Torulaspora, and Zygosaccharomyces sp. were detected only at the early stage. In addition, Cryptococcus, Microbotryum, Tetrapisispora, and Wickerhamomyces were detected as minor strains. Among the 62 compounds identified in this study, alcohols, ketones, and pyrazines were present as the major groups during the initial stages, whereas the abundance of acids with aldehydes increased as the fermentation progressed. Finally, the impacts of 10 different yeast strains found to participate in fermentation on the formation of volatile compounds were evaluated under soy‐based conditions. It was revealed that specific species produced different profiles of volatile compounds, some of which were significant flavor contributors, especially volatile alcohols, aldehydes, esters, and ketones.     

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.     

Quality evaluation of Korean soy sauce fermented in Korean earthenware (Onggi) with different glazes

The aim of this study was to investigate the effect of porosity‐controlled earthenware as fermentation vessels for Korean soy sauce. Porosity of fermentation vessels was controlled by glazing the surfaces of Korean earthenware. Three kinds of onggis– the outside glazed, inside and outside glazed, and unglazed onggi – were made and used to investigate the effect of glazing on the fermentation of soy sauce. During fermentation of soy sauce in porosity‐controlled earthenwares at 30 °C for 4 months, physical, chemical, microbiological and sensory quality attributes were monitored. Compared to other vessels, soy sauce fermented in onggi with both inside and outside surfaces glazed showed less water loss (10.7%), salt content (17.6%) and pH (pH4.4) after the fourth month. It also produced higher total acidity (1.43%), protease activity (810 μg mL^?1 min^?1) and microbiological changes that included total aerobic bacteria [4.3 log(cfu mL^?1)], lactic acid bacteria [3.8 log(cfu mL^?1)] and yeast [4.2 log(cfu mL^?1)]. The contents of total nucleotide (200–255 mg per 100 g sample) and free amino acids (4634–4848 mg per 100 g sample) in soy sauce were not consistent with glazing, which may be more affected by other factors, such as water loss, than the porosity of vessels. However, the percentage of glutamic acid among total free amino acids was 23.6% in onggi with both surfaces glazed, which was a little higher than the 21.9% in the outside glazed and 21.5% in the unglazed. These positive physicochemical and microbiological changes during fermentation in onggi with both sides glazed also resulted in higher sensory quality.     

Autolysis of Aspergillus oryzae Mycelium and Effect on Volatile Flavor Compounds of Soy Sauce

The autolyzed mycelia of Aspergillus oryzae are rich in proteins, nucleic acids, sugar, and other biomacromolecules, and are one of the main contributors to the flavor profile of commercially important fermented goods, including soy sauce and miso. We induced autolysis of the mycelia of A. oryzae over 1 to 10 d, and found that the maximum dissolved amounts of total protein and nucleic acid ratio accounted for 28.63% and 88.93%, respectively. The organic acid content, such as citric acid, tartaric acid, succinic acid, lactic acid, and acetic acid, initially increased and then decreased as autolysis progressed, corresponding to changes in pH levels. The main characteristic flavor compounds in soy sauce, namely, ethanol, 2‐phenylethanol, and 2‐methoxy‐4‐vinylphenol, were all detected in the autolysate. Subsequently, we tested the effect of adding mycelia of A. oryzae during the fermentation process of soy sauce for 60 d, and found that addition of 1.2‰ A. oryzae mycelia provided the richest flavor. Overall, our findings suggest that compounds found in the autolysate of A. oryzae may promote the flavor compounds of soy sauce, such as alcohols, aldehydes, phenols, and esters.     

Construction of the mutant strain in Aspergillus oryzae 3.042 for abundant proteinase production by the N+ ion implantation mutagenesis

Because of secreting large amounts of enzymes, Aspergillus oryzae was widely used in the fermentation of soy sauce in many Asian countries. Here, N⁺ ion implantation mutagenesis was applied to improve the ability of A. oryzae to secrete acid protease. Several mutants were screened using three kinds of plates (Casein medium, Czapek’s medium and carboxymethyl cellulose medium agar plates). Compared with the other mutants, the mutant A100‐8 could secrete the most protease. Acid protease activity of A100‐8 was enhanced about 44.1% at 36 h by koji fermentation test. In addition, A100‐8 was stable by periodically subculturing and maintaining on the agar slant. The mRNA expression levels of two kinds of acid protease (serine carboxypeptidase and aspartyl protease) were measured by RT‐PCR at different periods. Serine carboxypeptidase and some kinds of aspartyl protease of A100‐8 were significantly (P < 0.01) expressed higher than the control at all times.     

Effect of fermentation by Aspergillus oryzae on the biochemical and sensory properties of anchovy (Engraulis japonicus) fish sauce

This study obtained an anchovy flavourful fish sauce by a bioprocess of aerobic fermentation with Aspergillus oryzae OAY1 in a period of time as short as 3 days. Amino nitrogen of the fish sauce increased to 7.35 mg mL^?1 by optimisation with response surface methodology (RSM). Results of chemical analysis showed that amino nitrogen, hydrolysis degree, reducing sugar and total amino acids all increased, along with the enhanced antioxidant activity. With regard to biogenic amines, most of the tested biogenic amines decreased after fermentation. Electronic nose test revealed that the odour of the fish sauce became more balanced and favourable after fermentation. In addition, volatile odorant analysis and sensory evaluation showed that no particularly unpleasant flavour ingredient was found in the anchovy fish sauce, and the taste was overall satisfactory. Meanwhile, consumer acceptance test showed that the anchovy fish sauce produced in this study using A. orzyae OAY1 had a high consumer acceptance similar to the commercial anchovy fish sauce. This aerobic fast‐fermentation process provided an efficient method for high‐quality fish sauce production.     

The effect of the Corynebacterium glutamicum on the shortening of fermentation time,physicochemical and sensory properties of soy sauce

This study for the first time explores the possibility of using Corynebacterium glutamicum Ⅲ (C. glutamicum) to shorten the soy sauce fermentation period without compromising product quality. C. glutamicum was added to soy sauce that had been fermented with Aspergillus oryzae for 30 days (SS30) to initiate a further 3-day fermentation (yielding SS33). The 3-day fermentation exerted similar impacts on SS30 as the standard fermentation for further 60 days (yielding SS90): that is, increased the contents of total nitrogen, total free amino acids (FAAs), umami FAAs, acids, esters, ketones, furans and sulphur-containing compounds, whilst decreasing the contents of alcohols and phenols. This explained the similarities in taste, aroma and overall liking between SS33 and SS90. The SS90 was the darkest among the three soy sauces. Compared with SS90, SS33 was less salty and smoky, more umami and kokumi, softer and more mellow, higher overall liking, with more 0.5–1 kDa peptides and fewer peptides <0.5 kDa. Therefore, a 3-day fermentation with C. glutamicum after an initial 30 days of a normal soy sauce fermentation represents a simple and effective way to shorten the soy sauce production time from 90 to 33 days whilst increasing greatly the umami taste and mellow sensation. This approach enhances product competitiveness via lowering manufacturing cost.     

带鱼副产物鱼粉对酱醪品质的影响

目的:探讨在酱醪中添加带鱼鱼粉对酱醪品质的影响。方法:在舟山传统酱醪制备基础上添加带鱼鱼粉,测定酱油曲制备过程中酵母菌及米曲霉孢子数的变化,用福林酚法研究带鱼鱼粉对酱油曲蛋白酶活力的影响,采用GC-MS技术探讨带鱼鱼粉对发酵酱醪中挥发性成分的影响。结果:添加适量带鱼鱼粉对制曲过程中(36 h)的酵母菌和米曲霉孢子有一定促进效果,使各组孢子数均达到8.5×108以上;添加带鱼鱼粉后除发酵24 h外,其余发酵时间内各处理组的蛋白酶活力显著高于对照组(P<0.05);各试验组酱醪的氨基酸态氮在发酵30 d时,分别高于对照组14.5%,18.8%和19.4%;各试验组中检出挥发性成分33种,其中4%添加量组挥发性成分含量高于对照组11.55%;虽然低于6%添加量组,但壬醛等鱼腥味成分明显降低。结论:添加适量带鱼鱼粉对酱油曲中各微生物生长及蛋白酶活力具有一定促进作用,对酱醪的色泽、香气、流动性无明显影响,且可提高酱醪中氨基酸态氮的含量,添加鱼粉对酱醪挥发性成分种类基本没有影响,而鱼粉添加量过高会给酱醪带来不良风味。     

Effect of different halophilic microbial fermentation patterns on the volatile compound profiles and sensory properties of soy sauce moromi

The effect of inoculated or co-cultured halophilic lactic bacteria (Tetragenococcus halophilus) and yeasts (Zygosaccharomyces rouxii and Candida versatilis) on the quality of soy sauce moromi was investigated. Results showed no significant change in the major physicochemical properties of soy sauce moromi such as amino nitrogen, total acid, and reducing sugar at the end of fermentation, whereas the composition of free amino acids slightly changed with different fermentation patterns. In the sample inoculated with T. halophilus, the concentration of proline and tryptophan decreased by 18.83 and 50.87 % compared with the blank sample, whereas tyrosine and histidine increased by 32.41 and 27.04 %. In the sample inoculated with Z. rouxii and C. versatilis, the concentration of bitter-tasting lysine, tyrosine, and phenylalanine increased by more than 20 %, and the concentration of tryptophan decreased by 56.71 %. The proportion of amino acids with sweet taste increased by 0.8 % through the co-culture of the three strains. Meanwhile, the total amount of volatile compounds increased by 117.66, 23.28, and 216.88 % in the T. halophilus inoculated sample, two yeast strain inoculated sample, and three strain inoculated sample, respectively. Increased odour activity values of the nine compounds that mainly contributed to the flavour of moromi ranged from 1 to 7.87 times in the three strain inoculated sample. These findings revealed that the co-culture of the three strains during moromi fermentation was beneficial to the improvement of flavour of soy sauce moromi.     

多菌种发酵是提高酱油、食醋质量的重要途径

酿造酱油可采用2-3株曲霉菌复合制曲或分开制曲混合发酵，在发酵期添加鲁氏酵母、球拟酵母及少量乳酸菌，以提高酱油风味。酿造食酷在单一的黑曲霉(麸曲)中添加根霉、米曲霉或As3．350黑曲霉，中后期添加乳酸菌与酵母菌共酵，提高食醋中含氮量及增加不挥发酸的比例，并重视陈酿后熟阶段，改善食醋风味。提出应进一步研究传统大曲、麦曲、酱醪、醋醅，从中分离筛选出更多更好的有益菌种，并采用现代技术革新生产工艺，使酿造调味品生产高效、产品优质。     

低盐固态发酵酱油生产工艺的改进研究

该研究对低盐固态发酵酱油生产工艺进行了改进尝试,在制曲过程中添加富含各种矿物质的膨润土,既有利于霉菌的生长,增加产酶,又提高了酱油产品中的矿物质含量;采用双菌种制曲并在发酵后期添加增香酵母,进行淋浇。这样既改善了酱油的质量和风味,又使全氮利用率比对照组提高了5.13%,氨基酸生成率提高了2.75%。     

添加大曲发酵对广式酱油品质影响的研究

在酱油制曲及酱醪发酵阶段添加不同比例（0、2.75%、5.48%、8.17%）大曲,考察添加大曲对广式酱油制曲及酱油发酵品质的影响。结果表明,添加大曲对酱油成曲及酱油品质有影响,添加2.75%大曲的酱油主要理化指标氨基酸态氮（AAN）含量最高（0.87 g/100 mL）,感官评分最高（8.06分）,品质最优。微生物群落组成分析发现,添加大曲发酵的酱醪中四联球菌属（Tetragenococcus）和接合酵母属（Zygosaccharomyces）菌群相对丰度高于对照样,这可能是导致添加大曲酱油的总酸和酯类风味物质相对含量高于对照样酱油的原因。     

强化鲁氏接合酵母对酱油品质的影响

为了研究强化鲁氏接合酵母（JL-02）对酱油品质的影响。通过测定JL-02菌株耐盐性、成曲pH和中性酶活力、不同酿造工艺两种酱油的理化指标以及挥发性风味物质并进行分析。结果表明,JL-02菌株耐盐性高达16%,成曲pH为6.85±0.08,中性蛋白酶活力为（2109.2±45.2）U/g;强化鲁氏接合酵母后的酱油总酸含量为（1.14±0.01）g/100 mL,较对照组有所增加,而还原糖含量为（1.06±0.01）g/100 mL,pH为5.09±0.03,均较对照组有所降低,氨基态氮含量并无明显差异,均达到特级酱油标准。同时成曲、对照组和实验组挥发性风味物质分别为15、52和57种,而后者新增呋喃类风味物质（2-正戊基呋喃、4-甲氧基-2,5-二甲基-3(2H)-呋喃酮和2,3-二氢苯并呋喃）,对三组样品挥发性风味物质进行主成分分析,聚类分析结果良好。本研究为今后酱油企业进行强化酵母菌株酿造酱油工艺提供一定的思路和技术方法。     

Improvement of Fish Sauce Quality by Strain CMC5‐3‐1: A Novel Species of Staphylococcus sp.

Staphylococcus sp. CMC5‐3‐1 and CMS5‐7‐5 isolated from fermented fish sauce at 3 to 7 mo, respectively, showed different characteristics on protein hydrolysis and volatile formation. These Gram‐positive cocci were able to grow in up to 15% NaCl with the optimum at 0.5% to 5% NaCl in tryptic soy broth. Based on ribosomal 16S rRNA gene sequences, Staphylococcus sp. CMC5‐3‐1 and CMS5‐7‐5 showed 99.0% similarity to that of Staphylococcus piscifermentans JCM 6057^T, but DNA–DNA relatedness was <30%, indicating that they were likely to be new species. DNA relatedness between these 2 strains was only 65%, suggesting that they also belonged to different species. The α‐amino group content of 6‐month‐old fish sauce inoculated with Staphylococcus sp. CMC5‐3‐1 was 740.5 mM, which was higher than that inoculated by the strain CMS5‐7‐5 (662.14 mM, P < 0.05). Histamine was not produced during fermentations with both strains. Fish sauce inoculated with Staphylococcus sp. CMC5‐3‐1 showed the highest content of total glutamic acid (P < 0.05). The major volatile compound detected in fish sauce inoculated with Staphylococcus sp. CMC5‐3‐1 was 2‐methypropanal, contributing to the desirable dark chocolate note. Staphylococcus sp. CMC5‐3‐1 could be applied as a starter culture to improve the umami and aroma of fish sauce.     

发酵玉米皮渣制取酵母自溶液生产酱油的探讨

利用玉米提取淀粉和蛋白后的副产品玉米皮渣作为糖化曲的原料,再利用糖化曲水解玉米皮渣制取糖化液,补充氮源后制备液体酵母培养基,进而利用该培养基培养液体酵母,并且利用酵母自溶技术将其制成生产酱油用的酵母自溶液。结果表明:利用酵母自溶液配制的酱油,其全氮达到1.50g/100mL,氨基酸态氮达到0.78g/100mL,同时酱油的鲜味也得到了明显提高。