Effect of Tetragenococcus halophilus,Zygosaccharomyces rouxii,and Torulopsis versatilis addition sequence on soy sauce fermentation

The aim of this study was to investigate the effect that the sequences of ingredient addition had on soy sauce fermentation. Five strategies were assessed for the inoculation of Tetragenococcus halophilus, Zygosaccharomyces rouxii, and Torulopsis versatilis. The results of physicochemical analysis and levels of flavor compounds demonstrated that the best strategy for soy sauce fermentation was to inoculate T. halophilus (2 × 10⁵ CFU/mL) on the 15th day, Z. rouxii (1.0 × 10⁶ CFU/mL) on the 30th day, and T. versatilis (1.0 × 10⁶ CFU/mL) on the 45th day; this enhanced its fruity, sauce, alcoholic, and caramel-like flavors by 64.3%, 22.7%, 43.1%, and 36.2%, respectively, and saline taste by 64.3%. Spearman correlation analysis revealed the content of ethanol positively correlated with acetic acid (r = 0.82) and succinic acid (r = 0.80), while the level of formic acid negatively correlated with ethyl phenylacetate (r = −0.88). In summary, these results provided fundamental evidence for strain addition sequence for producing flavor-enhanced soy sauce with potential application in the future.     

Microbiota during fermentation of chum salmon (Oncorhynchus keta) sauce mash inoculated with halotolerant microbial starters: Analyses using the plate count method and PCR-denaturing gradient gel electrophoresis (DGGE)

Nine different combinations of mugi koji (barley steamed and molded with Aspergillus oryzae) and halotolerant microorganisms (HTMs), Zygosaccharomyces rouxii, Candida versatilis, and Tetragenococcus halophilus, were inoculated into chum salmon sauce mash under a non-aseptic condition used in industrial fish sauce production and fermented at 35 ± 2.5 °C for 84 days to elucidate the microbial dynamics (i.e., microbial count and microbiota) during fermentation. The viable count of halotolerant yeast (HTY) in fermented chum salmon sauce (FCSS) mash showed various time courses dependent on the combination of the starter microorganisms. Halotolerant lactic acid bacteria (HTL) were detected morphologically and physiologically only from FCSS mash inoculated with T. halophilus alone or with T. halophilus and C. versatilis during the first 28 days of fermentation. Only four fungal species, Z. rouxii, C. versatilis, Pichia guilliermondii, and A. oryzae, were detected throughout the fermentation by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In FCSS mash, dominant HTMs, especially eumycetes, were nonexistent. However, under the non-aseptic conditions, undesirable wild yeast such as P. guilliermondii grew fortuitously. Therefore, HTY inoculation into FCSS mash at the beginning of fermentation is effective in preventing the growth of wild yeast and the resultant unfavorable flavor.     

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.     

Genome shuffling of Zygosaccharomyces rouxii to accelerate and enhance the flavour formation of soy sauce

BACKGROUND: The purpose of this study was to achieve rapid improvement of the flavour of soy sauce by increasing the salt stress resistance of Zygosaccharomyces rouxii. Here, we describe genome shuffling to improve the salt tolerance of Z. rouxii while simultaneously enhancing and accelerating flavour formation of soy sauce. RESULTS: A mutant, S3‐2, with a stronger resistance to salt, was selected after three rounds of genome shuffling. S3‐2 not only grew well in peptone/yeast extract/dextrose medium containing a high salt content with wide range of pH, but also exhibited stronger stress resistance to potassium chloride and lithium chloride. In high‐salt liquid fermentation, S3‐2 obviously accelerated flavour formation of soy sauce, thus decreasing the total time required for development of the aroma. In addition, S3‐2 gave high amino acid nitrogen and good flavour. In particular, the ethyl acetate content was 2.38 times that in the control. S3‐2 distinctly improved the formation of 4‐hydroxy‐2 (or 5) ‐ethyl‐5 (or 2) ‐methyl‐3 (2H) ‐furanone by up to 75%. Another important flavour component, 4‐ethylguaiacol, was also detected. CONCLUSIONS: Genome shuffling was successfully used to achieve significant improvements in flavour formation. The selected strain improved the main flavour components and amino acid nitrogen, thereby enhancing the quality of soy sauce. Copyright © 2009 Society of Chemical Industry     

Comparing the differences of characteristic flavour between natural maturation and starter culture for Mucor‐type Douchi

In this research, effects of maturation times on volatiles of Mucor‐type Douchi were investigated. Results showed that 2‐year matured sample exhibited higher concentrations of characteristic flavours including alcohols, esters, phenols, ketones and pyrazines. Additionally, effects of starter cultures addition on volatiles were investigated, and results demonstrated that inoculated fermented samples presented higher amounts of aldehydes, ketones, phenols and pyrazines compared with that of naturally fermented sample. Analysis of odour activity value (OAV) suggested that increased OAVs of eight compounds that mainly contributed to the flavour of Douchi ranged from onefold to 6.5‐fold in the Tetragenococcus halophilus, Zygosaccharomyces rouxii and Candida versatilis co‐cultured sample. Furtherly, by comparing the characteristic flavour of co‐cultured and 2‐year matured samples, no significant difference was observed in the OAV of flavour except rose‐like and soapy flavour, which indicated that co‐culture of the three strains during Douchi fermentation may facilitate the formation of flavours and shorten the fermentation process.     

Fermentation of Soy Sauce with Immobilized Whole Cells

Soy sauce was produced continuously for 80 days in 280 liter column-type reactors containing immobilized whole cells of Pediococcus halophilus, Saccharomyces rouxii, and Torulopsis versatilis entrapped in calcium alginate gels. The divided lactic acid and alcohol fermentation by viable cells of three kinds with feed solution obtained from enzymatic hydrolyzate of koji and defatted soybean meal proceeded in shorter time than a complex fermentation of moromi (soy sauce mash) in the slurry state. The refined products fermented by this process had good taste and flavor, and were close to the conventional soy sauce with respect to organic acid and aroma components.     

耐盐米曲霉CICIM F0899在酱油酿造过程中的应用

耐高盐环境的蛋白酶对酱油工业有着积极作用。该实验对1株产耐盐蛋白酶的米曲霉CICIM F0899在高盐稀态酱油发酵过程中的作用进行了研究,并同沪酿3042进行了比较。结果表明,在酱醪发酵阶段,微生物代谢产酸使酱醪pH持续下降,影响了蛋白酶的活性,导致酱油中氨基态氮和风味物质含量的变化,在180天时氨基态氮含量达到0.62 g/100 mL,比沪酿3042高11.11%。利用气相色谱-质谱联用(GC-MS)对挥发性成分进行了分析鉴定,检测出酱醪中7大类共49种挥发性化合物,其中酯类、杂环类化合物的含量明显高于沪酿3042,而醛类物质的含量相对较少。因此,米曲霉CICIM F0899在氨基态氮含量和风味组成方面都具有更加明显的优势。     

耐盐乳酸菌和酵母菌强化技术对高盐稀态发酵酱醪品质的影响

分别将耐盐乳酸菌Tetragenococcus halophilus CGMCC 3792、耐盐酵母菌Zygosaccharomyces rouxii CGM-CC 3791和Candida versatilis CGMCC 3790菌悬液接入酱醪中,利用顶空-固相微萃取偶联气质联用技术(HS-SPME/GC-MS)检测了发酵125 d酱醪中挥发性组分的变化。研究结果表明,T.halophilus CGMCC 3792强化的酱醪中酸类、醇类、酮类、呋喃类、吡嗪类和其他组分的相对含量降低,酯类、醛类和酚类组分的相对含量增加,其中苯乙醇、乙酸乙酯、辛酸乙酯、癸酸乙酯和4-乙基愈创木酚等的相对含量显著增加,而Z.rouxii CGMCC 3791、C.versatilis CGMCC 3790共培养强化的酱醪中醇类、酯类、醛类和其他组分的相对含量增加,酸类、酚类、呋喃类和吡嗪类组分的相对含量降低,其中苯乙醇、长链脂肪酸乙酯(月桂酸乙酯、棕榈酸乙酯、亚油酸乙酯等)的相对含量显著增加。此外,本研究中T.halophilus CGMCC 3792强化的酱醪中4-乙基愈创木酚的相对含量大幅增加,且在整个发酵过程中其含量也单调增加,对于T.halophilus CGMCC 3792是否具有分泌4-乙基愈创木酚的能力以及是否冷温工艺赋予该菌株合成4-乙基愈创木酚的能力需进一步探究。     

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.

     

促酱醪发酵芽孢杆菌的筛选及应用

用酪素平板法从高盐稀态酱醪中筛选出56 株蛋白酶活性较好的菌株,牛津杯法复筛后制曲测定酶活性,得到淀粉酶活性最高的菌株CS1.11、蛋白酶活性最高的菌株CS1.13及纤维素酶活性最高的菌株CS1.17;16S rRNA序列测定结合形态学分析,CS1.11、CS1.13、CS1.17分别鉴定为贝莱斯芽孢杆菌（Bacillus velezensis）、甲基营养型芽孢杆菌（B. methylotrophicus）、枯草芽孢杆菌（B. subtilis）;分别将3 株实验菌与对照菌枯草芽孢杆菌CS1.03单独进行制曲后盐卤发酵,发现CS1.13发酵酱油氨态氮质量浓度最高,为5.12 g/L,CS1.11发酵酱油还原糖质量浓度最高,为27.20 g/L,与其酶活性结果一致,4 株菌对酱油总酸含量影响不大;顶空固相微萃取气相色谱-质谱联用内标法定性及定量分析发酵酱油发现,3 株实验菌与对照菌均具有产生吡嗪类物质及其前体乙偶姻和2,3-丁二醇的优势,且3 株实验菌优于对照菌;吡嗪类物质能赋予酱油良好的风味及健康因子,是芽孢杆菌酱油发酵的特征风味物质。筛选自高盐稀态酱醪的芽孢杆菌CS1.11、CS1.13、CS1.17有促酱醪发酵、丰富酱油风味、增进健康因子的潜力,具备良好的应用前景。     

Production of tyramine in "moromi" mash during soy sauce fermentation

The concentrations of 7 non-volatile amines, tyramine (Tym), histamine (Him), phenethylamine (Phm), putrescine (Put), cadaverine (Cad), spermidine (Spd) and spermine (Spm) in the liquid part of "moromi" mash during soy sauce fermentation were studied. These amines, except for him and Cad, were detected during fermentation by the conventional production method in the laboratory. Put and Spd were detected at the beginning, and Tym, Phm and Spm appeared later; these 5 amines increased gradually during the fermentation. Put, Spd, Spm and Cad were present in the raw starting material for soy sauce; thus, Tym and Phm were produced by the fermentation. When "moromi" mash was added to liquid medium and cultivated, Tym was detected in some "moromi" mash and the other amines were not detected. Tym-producing bacterial strains were isolated from the liquid culture media of Tym-positive "moromi" mash. The Tym-producing strain was a gram-positive coccus. The conditions for production of amines by Tym-producing bacterial strains were examined. These strains grew and produced tyramine under various conditions, which may occur during soy sauce fermentation. Namely, Tym was produced at pH 5-10, at salt concentrations of less than 8%, under either aerobic or anaerobic conditions. During soy sauce fermentation, it is assumed that Tym would be produced by these strains during the early stages of soy sauce aging within a short period when the salt concentration and pH conditions are optimal for growth. Based on the bacteriological properties, the strains were identified as Enterococcus faecium. With the exception of Phm and Him, which did not exist in the starting raw material, non-volatile amines (including Put, Cad, Spd and Spm) were not produced and microorganisms producing them are not believed to be present during "moromi" fermentation.     

Fermentation technology and quality of sauce prepared from rice dreg protein

Fermented sauce, from rice dreg protein (RDP) instead of soybean, has unique advantages. In the present paper, the fermentation technology of RDP sauce was studied. Amino nitrogen, reducing sugar, soluble protein and colour indicators, as well as the quality of three kinds of RDP sauce fermented under different conditions, were analysed. The results suggest that an optimized fermentation technology is the addition of 2.4 times salty solution into koji, to obtain a 13% salt concentration in the sauce mash, and then fermentation at ~35°C. RDP sauce that had an addition of 0.01% Zygosaccharomyces rouxii in the fermentation stage gave a good quality RDP sauce. The RDP sauce content of amino nitrogen and reducing sugar, with 0.01% Z. rouxii, was 0.98 and 4.57 g/100 mL respectively, and the corresponding sensory quality was better than in the samples produced without Z. rouxii. It was concluded that RDP can be utilized to produce a high‐quality sauce. Copyright © 2015 The Institute of Brewing & Distilling     

利用富硒双孢菇粉提升酱油有机硒含量和改善酱油风味

该研究利用临期和损伤富硒双孢菇制备富硒双孢菇粉,以其替代15%质量分数的面粉,按照高盐稀态工艺生产富硒酱油（样品酱油）。结果显示,样品酱油总硒和有机硒含量分别达到134.65和122.53μg/kg,比对照酱油提高5.70倍和5.85倍,有机硒转化率为91.00%,符合富硒酱油标准。理化分析结果表明样品酱油总氮、氨基酸态氮和无盐固形物含量为1.72、1.03和22.56 g/100 mL,比对照酱油提高9.55%、19.77%和5.13%。此外,样品酱油蘑菇香、麦芽香、醇香和果香香气强度比对照酱油提高了5 837.17%、15.62%、20.15%和15.88%。添加富硒双孢菇粉显著提高了大曲和酱醪中蛋白酶酶活,这是富硒双孢菇粉提高酱油滋味物质含量的原因。富硒双孢菇粉中的1-辛烯-3-酮、1-辛烯-3-醇、3-辛醇和3-辛酮赋予了样品酱油蘑菇香。此外,富硒双孢菇粉促进了酱醪中产香微生物的繁殖,进而提高了样品酱油中麦芽香、醇香和果香香气强度。该研究利用临期和损伤富硒双孢菇开发了一种风味良好的富硒酱油,可为双孢菇和酱油生产企业提高产品附加值提供技术参考。     

Evaluation of a Malaysian soy sauce <Emphasis Type="Italic">koji</Emphasis> strain <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> NSK for γ-aminobutyric acid (GABA) production using different native sugars

In this study, a selected γ-aminobutyric acid (GABA)-rich Malaysian strain Aspergillus oryzae NSK was collected from soy sauce koji. The strain was used to explore the effect of using renewable native sugar syrup, sugarcane, nipa, and molasses as fermentable substrates for developing a novel functional GABA soy sauce. We evaluated the strain using the chosen native sugars for 7 days using shake flask fermentation at 30 °C. The results showed optimum GABA concentration was achieved using cane molasses as the fermentable substrate (354.08 mg/L), followed by sugarcane syrup (320.7 mg/L) and nipa syrup (232.07 mg/L). Cane molasses was subsequently utilized as a substrate to determine the most suitable concentration for A. oryzae NSK to enhance GABA production and was determined as 50% g/L of glucose standard cane molasses. Our findings indicate that cane molasses can be used as a GABA-rich ingredient to develop a new starter culture for A. oryzae NSK soy sauce production.     

Effect of temperature on the stability of various peptidases during peptide-enriched soy sauce fermentation

We previously developed a peptide-enriched soy sauce-like seasoning called Fermented Soybean Seasoning (FSS) with high-temperature fermentation, and we have reported the antihypertensive effects of FSS. Seryl-tyrosine (Ser-Tyr) and glycyl-tyrosine (Gly-Tyr) were identified from FSS as active constituents in the antihypertensive effects. They were found to be particularly enriched in FSS; more so than in regular soy sauce. In the present study, we clarified one of the mechanisms underlying the accumulation of these bioactive peptides during high temperature soy sauce brewing. Crude enzyme extracts were prepared from model soy sauce mash (moromi) fermented at various temperatures. Leucine aminopeptidase-I, II, and seryl-tyrosine hydrolytic activity were found to decrease in the moromi incubated at the fermentation temperature of FSS whereas almost no decrease was observed in that of regular soy sauce. The concentrations of ACE inhibitory peptides, Ser-Tyr and Gly-Tyr, in the moromi incubated at high temperature were revealed to be higher than those at low temperature through quantitative LC-MS/MS analysis. These results suggested that the peptidases responsible for degrading low molecular weight bioactive peptides were inactivated during the high temperature fermentation, thus, these peptides would be likely to remain in the high temperature fermentation.     

糖化增香曲对高盐稀态酱油发酵过程中理化性质影响的研究

本文通过添加糖化增香曲混合制曲(KG)生产高盐稀态酱油,对比纯种米曲霉制曲(KP),研究糖化增香曲对高盐稀态酱油发酵过程中理化性质的影响.研究结果表明:添加糖化增香曲混合制曲可显著提高高盐稀态酱油的还原糖和可溶性无盐固形物含量,发酵90 d时分别较纯种制曲提高47%和7%:也可显著提高发酵初期曲料中各类物质的溶出速率;...     

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.     

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.     

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.     

Proteinase-producing halophilic lactic acid bacteria isolated from fish sauce fermentation and their ability to produce volatile compounds

Halophilic lactic acid bacteria were isolated from fish sauce mashes fermented at 1 to 12 months. Seven out of sixty-four isolates were selected according to their proteolytic activity and growth at 25% NaCl for characterization and investigation of volatile compound production. All selected isolates were Gram-positive cocci with pairs/tetrads and grew at 0-25% NaCl, pH 4.5-9.0. Results of 16S rRNA gene sequence analysis showed 99% homology to Tetragenococcus halophilus ATCC 33315. The restriction fragment length polymorphism (RFLP) patterns of all isolates were also similar to those of T. halophilus ATCC 33315. These isolates were, thus, identified as T. halophilus. All isolates hydrolyzed fish protein in the medium containing 25% NaCl. Intracellular aminopeptidase of 7 isolates exhibited the highest activity of 2.85-3.67 U/ml toward Ala-p-nitroanilide (Ala-pNA). T.halophilus strains MS33 and M11 showed the highest alanyl aminopeptidase activity (P < 0.05), and produced histamine in mGYP broth containing 5 and 25% NaCl in the level of 6.62-22.55 and 13.14-20.39 mg/100 ml, respectively. Predominant volatile compounds of fish broth containing 25% NaCl inoculated with T. halophilus MS33 and MRC5-5-2 were 1-propanol, 2-methylpropanal, and benzaldehyde, corresponding to major volatile compounds in fish sauce. T.halophilus appeared to play an important role in volatile compound formation during fish sauce fermentation.