解淀粉芽孢杆菌SWJS22和米曲霉混合制曲在酱油发酵中的应用

以解淀粉芽孢杆菌SWJS22和米曲霉为发酵菌株,考察米曲霉单独制曲发酵、米曲酶和解淀粉芽孢杆菌曲料混合发酵以及双菌种混合制曲发酵对酱油理化指标及风味的影响。结果表明:曲料混合发酵的酱油氨基酸转化率有较大提高,色率和色深物质均低于米曲霉单独制曲发酵的酱油和双菌种混合制曲发酵的酱油,曲料混合发酵酱油和双菌种混合制曲发酵的酱油谷氨酸含量有明显提高。采用气相色谱-质谱对比分析3种不同曲料发酵酱油的风味物质,结果表明:3种酱油的风味物质种类差别不明显,重要风味成分在3种酱油中均有检出,曲料混合发酵的酱油酸类物质的含量最低。     

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。     

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.     

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 a halophilic aromatic yeast together with Aspergillus oryzae in koji making on the volatile compounds and quality of soy sauce moromi

Koji making is an essential step in the production of high‐quality soy sauce. In this study, we inoculated koji with a halophilic aromatic yeast (Zygosaccharomyces rouxii) and Aspergillus oryzae and investigated the effects on the volatile compounds and quality of soy sauce moromi. Our results suggest that the optimal yeast inoculation was approximately 1.5 × 10⁶ yeast per gram of koji, and excessive yeast greatly decreased A. oryzae enzyme activity. In comparison with the control group, there were no significant changes in the concentrations of amino nitrogen, total titratable acids, total soluble nitrogen and reducing sugar by extending the fermentation time during the first fermentation month, whereas the concentration of glutamate in the yeast‐inoculated sample increased by 46.16% and the arginine concentration decreased by 61.07%. After the second fermentation month, 17, 38 and 38 volatile components were identified by GC–MS in C1, C2 and C5, respectively. Moreover, a large number of volatile components, such as 2‐Methoxy‐4‐vinylphenol, ethyl acetate and 1‐Octen‐3‐ol were found in C2 and C5. And the total contents of volatile compounds in C2 were obviously higher than C5. This work furthers our understanding of the traditional multistrain koji making and provides a new method for enhancing the quality and flavour of soy sauce.     

非耐盐乳酸菌与米曲霉共生酿造酱油的研究

文章将米曲霉与非耐盐的乳酸菌——融合魏斯氏菌(Weissella confusa)共同制曲,研究了其对制曲过程中主要酶系活力和发酵过程中质量指标、酚类物质和抗氧化活性等的影响,结果显示:酸性蛋白酶、纤维素酶、果胶酶、β-葡萄糖苷酶的活力较米曲霉组有显著提高,而中性蛋白酶活和α-淀粉酶活没有显著变化。添加了乳酸菌后的酱油不仅提高了酱油中氨基酸态氮、总氮、还原糖的含量,同时酱油中总多酚和总黄酮含量及自由基清除能力都显著增加,而有机酸含量变化较小。     

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.     

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.     

酱油生产中应用米曲霉和黑曲霉混合制曲的探索

该文考察了米曲霉和黑曲霉在单独制曲及混合制曲条件下,成曲的中性蛋白酶、酸性蛋白酶、糖化酶及孢子数,探讨了酱油多菌种制曲的可行性.实验以豆粕、麸皮为原料,以AS3.042米曲霉、AS3.350黑曲霉为菌种,探讨了2种制曲方式及不同配比制曲效果的影响.结果表明,米、黑曲霉分开制曲比混合接种制曲所得成曲效果好,米、黑曲霉分别以33℃制曲42h、48h得成品曲,再以2∶1混合所得成曲可获得较佳的酶活力,曲料总蛋白酶活力2748U/g与对照值相当,而酸性蛋白酶及糖化酶活力分别达712U/g、1090U/g,较对照分别提高55.8％、25.4％.     

双孢菇酱油双酿工艺研究

以双孢菇及豆粕、小麦、麸皮等为原料,通过米曲霉和鲁氏酵母的混合种曲在28℃～30℃发酵40d,将原料中蛋白质降解,从而酿制出双孢菇酱油。该产品具有红褐色、酱香浓郁、菇香味突出、口感鲜美、咸淡适口等特征,其理化指标均达到酿造酱油一级的标准,为双孢菇的利用提供了新的思路。     

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.     

QUALITY OF FISH SAUCE PRODUCTS FROM RECYCLED BY‐PRODUCTS FROM FISH GEL AND KAMABOKO PROCESSING

ABSTRACT

In order to make effective reuse of kamaboko processing wastes and to minimize the amount of waste disposal, the production of two kinds of fish sauces was tried on a small industrial scale from the wastes with or without the addition of the meat of deepsea smelt (Glossanodon semifasciatus). They were fermented for 6 months at room temperature using salt and koji mold. As a control, a fish sauce was also produced only from the deepsea smelt meat. The recovery of fish sauce from the initial mashed mixture ranged between 75 and 79%, depending on the ingredients in the three fish sauce products. The total nitrogen content of the waste sauce and the mixed sauce was lower than that of the control. The levels of the original additives to kamaboko products, β‐carotene and sorbic acid, were very low in these fish sauces. The taste‐active components of the waste sauce and mixed sauces were lower than those of the control. Sensory evaluation revealed that the former two waste sauces were less bitter and higher in saltiness than the control. However, no difference was found in umami taste between these products. These findings suggest that the wastes from kamaboko processing factories could be reused as fish sauce for food condiments.

PRACTICAL APPLICATIONS

Recent development in the food industry in Japan has enabled the surimi‐based products to be mass produced and standardized even in the fish gel, kamaboko trade. However, a serious problem has arisen in the development of kamaboko processing through the discharge of the wastes, i.e., nonstandard products or fragments, from kamaboko processing factories. It is clear that the waste from kamaboko processing factories is transformed effectively into fish sauce by using soy sauce koji mold. As a result, the amount of the discharged wastes from kamaboko factories are able to be minimized because the liquefaction ratio of the fish sauce mushes (moromi) from the wastes after fermentation was high and the products have a high umami taste and agreeable soy sauce‐like flavor.     

Selection of micro-organisms for use in the fermentation of soy sauce

Micro-organisms related to soy sauce production are mainly koji mold (Aspergillus oryzae or Aspergillus sojae), lactic acid bacteria (Pediococcus halophilus) and osmophilic yeasts (Saccharomyces rouxii, Candida (Torulopsis) versatilis, Candida (Torulopsis) etchellsii).The selection parameters of these micro-organisms for use in soy sauce production are as follows.

1. (1) Aspergillus oryzae or Aspergillus sojae. (a) Do not produce mycotoxins; (b) possess high proteolytic, amylolytic, and macerating enzyme levels.

2. (2) Pediococcus halophilus. (a) Produce lactic acid and other useful organic acids in a high concentration of salt brine; (b) do not possess tyrosine decarboxylase and histidine decarboxylase.

3. (3) Saccharomyces rouxii. Produce alcohol and excellent flavor substances in a high salt concentration brine.

4. (4) Candida (Torulopsis) versatilis and C. etchellsii. Produce excellent flavor substances in a high salt concentration brine.

     

米渣生酱油和大豆生酱油的风味表征及比较

对3种不同菌种发酵的米渣生酱油和一种大豆生酱油的风味进行比较,以阐释鲁氏酵母、植物乳杆菌参与发酵对米渣生酱油风味的影响以及米渣生酱油和大豆生酱油的风味差别。结果表明单菌种发酵的大豆生酱油及鲁氏酵母和植物乳杆菌分别参与发酵的米渣生酱油游离氨基酸含量、味道强度值、挥发性风味成分及相对含量均明显高于米曲霉单菌种发酵的米渣生酱油,鲁氏酵母参与发酵的米渣生酱油中4-乙烯基愈创木酚相对含量达9.71%;电子舌呈味分析表明4种生酱油的主成分存在明显差异,鲁氏酵母参与发酵的米渣生酱油和大豆生酱油呈味非常相似,鲜味突出而酸味相对较弱,植物乳杆菌参与发酵的米渣生酱油各种味道相对均衡,综合感官质量最好。米渣酱油和大豆酱油的整体风味有一定差别,优化菌种耦合发酵明显有利于增强米渣生酱油风味。     

Investigation of the microbial changes during koji‐making process of Douchi by culture‐dependent techniques and PCR‐DGGE

Douchi as a traditional fermentation soyfood with an acquired taste had been appreciated by consumers for thousands of years in China and few Asian countries. Unfortunately, few studies had been conducted on the changes of microbial community during the koji‐making process, and whether the pathogenic microorganisms involving the fermentation process is still unclear. Therefore, the PCR‐denaturing gradient gel electrophoresis (DGGE) targeting the 16S and 18S rRNA genes was applied to characterise the dynamic changes of Bacteria, Bacilli and Fungi during koji‐making process. The results of DGGE showed that eleven species of bacteria, nine species of bacilli and seven species of Fungi had been detected during the koji‐making progress, of which the Bacillus subtilis and Aspergillus oryzae belonged to the dominant microorganisms. Also, eleven strains were isolated from Koji‐making samples and were identified as B. subtilis, Bacillus amyloliquefaciens, A. oryzae, Brachybacterium sp., Aspergillus niger, Staphylococcus saprophyticus, Micrococcus sp., Staphylococcus gallinarum, Absidia corymbifera, Saccharomyces cerevisiae, Malassezia sp.; Our results revealed that pathogenic microorganisms were involved in the koji‐making process, but the probiotic microorganisms occupied the dominant position of community in the koji‐making process.     

外加营养物对酱油种曲孢子数和成曲酶活力的影响

以提高酱油种曲孢子数和成曲酶活力为目标,在麸皮培养基的基础上,对酱油种曲培养基的外加碳源、氮源、无机盐进行优化研究。结果表明：在葡萄糖1.00%、氯化钙0.10%、硫酸锌0.05%、豆粕3.00% 的条件下孢子数可达(15.77 ± 0.49)× 109 个/g 干重,比对照组提高了84.87%。同时对优化种曲和利用该种曲制得的大曲的酶活力进行比较,表明种曲培养基的优劣对酱油发酵有重要影响,其中种曲酶活力提高25.04%,大曲酶活力提高17.40%。     

Development of the Rhodiola rosea Fuqu and Rhodiola rosea soy sauce,and the determination of their functional properties

A mixed starter, composed of high‐quality Rhodiola rosea Fuqu, was developed. Compared with normal Fuqu, Rhodiola rosea Fuqu demonstrated higher spore numbers, spore germination rates, protease activity, liquefying amylase activity and total acid and amino acid nitrogen. Specifically, the spore number was 1.36 times that of regular Fuqu. Protease activity, liquefying amylase activity, total acid and amino acid nitrogen were 0.475 g/100 mL, 104.16 U/g dry Qu, 35.67 mmol/L and 26.08 g/100 mL, respectively. These values were 21.48, 7.41, 6.07 and 67.82% higher than the regular Fuqu. The Rhodiola rosea soy sauce indices were also better than those for the control. The DPPH scavenging abilities of R. rosea soy sauce, control soy sauce, market soy sauce 1 and market soy sauce 2 were 74.25, 44.01, 34.73 and 11.38%, respectively. The total phenolic content in these four samples was 387.38, 309.23, 212.85 and 202.60 mg GAE/g, respectively. As observed with the DPPH and total phenolic assay, the R. rosea soy sauce showed the highest antioxidant capacity. A simple, rapid, and sensitive determination method of salidroside in R. rosea Fuqu and R. rosea soy sauce using HPLC‐ESI‐MS/MS was developed and validated with multiple reaction monitoring in the negative mode.     

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.     

Making soy sauce using direct fermentation of defatted soybean meal without the meju (soybean Koji) preparation process

Soy sauce prepared via direct fermentation of defatted soybean meal (DFSM) using halophiles without addition of dried, fermented soybeans or meju was evaluated. DFSM was fermented using single and mixed cultures of Oceanobacillus kimchii and Bacillus pumilus under 18% salinity conditions. Amounts of total organic nitrogen, free amino acids, and organic acids in soy sauce prepared with the mixed culture were slightly higher than sauces prepared with single culture. The ingredient content was higher in soy sauce prepared via direct fermentation of DFSM than soy sauce prepared with meju. Microorganisms detected in DFSM fermentation were not detected in the meju culture, except for the 2 halophiles, based on metagenomic analysis. Direct fermentation of DFSM is better than using meju for preparation of soy sauce.