Moromi Fermentation Research Articles

Characteristics Of Moromi Sweet Sauce And The Study Of Its Activity On Protein, Sugar And Viscosity Levels

The aim of this research was to determine the effect of moromi fermentation on soluble protein content, sugar content and viscosity, with varying salt treatments for 28 days. as well as organoleptic tests. The research design used a completely randomized design (CRD) consisting of 4 treatments and 6 replications. The treatment included fermenting black soybeans by A sojae (Koji) then soaking them in 1000 ml of water with the addition of 0%, 10%, 20% and 30% salt for 28 days (moromi). The parameters analyzed are protein content, sugar content and viscosity and organoleptic tests include color, aroma and taste. The research results showed that the highest protein content was found in the K1 treatment at 1.522%, the sugar content in K1 was 41.227% and the viscosity in K3 was 33.603%. Meanwhile, in terms of organoleptic color, aroma and taste, the panelists liked the product.

Developing a High-Umami, Low-Salt Soy Sauce through Accelerated Moromi Fermentation with Corynebacterium and Lactiplantibacillus Strains.

The traditional fermentation process of soy sauce employs a hyperhaline model and has a long fermentation period. A hyperhaline model can improve fermentation speed, but easily leads to the contamination of miscellaneous bacteria and fermentation failure. In this study, after the conventional koji and moromi fermentation, the fermentation broth was pasteurized and diluted, and then inoculated with three selected microorganisms including Corynebacterium glutamicum, Corynebacterium ammoniagenes, and Lactiplantibacillus plantarum for secondary fermentation. During this ten-day fermentation, the pH, free amino acids, organic acids, nucleotide acids, fatty acids, and volatile compounds were analyzed. The fermentation group inoculated with C. glutamicum accumulated the high content of amino acid nitrogen of 0.92 g/100 mL and glutamic acid of 509.4 mg/100 mL. The C. ammoniagenes group and L. plantarum group were rich in nucleotide and organic acid, respectively. The fermentation group inoculated with three microorganisms exhibited the best sensory attributes, showing the potential to develop a suitable fermentation method. The brewing speed of the proposed process in this study was faster than that of the traditional method, and the umami substances could be significantly accumulated in this low-salt fermented model (7% w/v NaCl). This study provides a reference for the low-salt and rapid fermentation of seasoning.

Proteomics analysis of enzyme systems and pathway changes during the moromi fermentation of soy sauce mash.

During the brewing of soy sauce, the conversion of multiple substances is driven by various microorganisms and their secreted enzyme systems. Soy sauce mash is an important source of enzyme systems during moromi fermentation, but the changes of enzyme systems in soy sauce mash during moromi fermentation are poorly understood. In order to explore the predominant enzyme systems existing during moromi fermentation and to explain the characteristics of the enzyme system changes, an enzymatic activities assay and 4D-label-free proteomics analysis were conducted on soy sauce mash at different stages of fermentation. The activities of hydrolytic enzymes in soy sauce mash decreased continuously throughout the fermentation process, while most of the characteristic physicochemical substances in soy sauce mash supernatant had already accumulated at the early stage of fermentation. Four hydrolytic enzymes were found to be positively correlated with important physicochemical indexes by principal component analysis and Pearson correlation analysis. The proteomics analysis revealed three highly upregulated enzymes and two enzymes that were present in important metabolic pathways throughout the fermentation process. Furthermore, it was found that Aspergillus oryzae was able to accumulate various nutrients in the soy sauce mash by downregulating most of its metabolic pathways. Enzymes present with excellent properties during the moromi fermentation period could be obtained from these results. Meanwhile, the characterization of the metabolic pathways of microorganisms during the moromi fermentation period was revealed. The results provide a basis for more scientific and purposeful improvement of moromi fermentation in the future. © 2024 Society of Chemical Industry.

Co‐culture relationship between Zygosaccharomyces rouxii and Candida versatilis and its effect on the flavour of soy sauce

SummaryZygosaccharomyces rouxii and Candida versatilis are important flavour‐producing yeasts in soy sauce fermentation. In the present study, the interaction between Z. rouxii and C. versatilis co‐cultures in YPD and raw soy sauce medium was investigated and applied to soy sauce fermentation. The concentration of Z. rouxii in the co‐culture was 0.47 times that in the mono‐culture. This observation suggested that C. versatilis inhibited the growth of Z. rouxii, likely attributed to various factors, including the production of acids and heat‐ and protease‐sensitive substances by C. versatilis. Furthermore, we examined the physicochemical properties and characteristic volatile flavour components of soy sauce by adding varying ratios of Z. rouxii and C. versatilis at different stages of fermentation. The results showed that mixed fermentation of Z. rouxii and C. versatilis enhanced the total nitrogen, amino acid nitrogen, 4‐hydroxy‐2(5)‐ethyl‐5(2)‐methyl‐3(2H)‐furanone, and volatile compounds in soy sauce compared to single‐strain fermentation. Additionally, optimal soy sauce results were obtained when yeasts were added on day 45, with an inoculation ratio of 1:1. In conclusion, during the moromi fermentation stage, the appropriate selection of the inoculation ratio and time for Z. rouxii and C. versatilis addition can effectively enhance the physicochemical properties and flavour attributes of soy sauce.

Effect of Sequential Inoculation of Tetragenococcus halophilus and Wickerhamomyces anomalus on the Flavour Formation of Early-Stage Moromi Fermented at a Lower Temperature.

Microbial inoculation in moromi fermentation has a great influence on the physicochemical and flavour properties of soy sauces. This work investigated the effect of inoculating Tetragenococcus halophilus and Wickerhamomyces anomalus on the flavour formation of early-stage moromi (30 days) fermented at a lower temperature (22 °C) by determining their physicochemical and aroma changes. The results showed that single yeast or LAB inoculation increased the production of amino nitrogen, lactic acid and acetic acid, as well as free amino acids and key flavour components. Particularly, the sequential inoculation of T. halophilus and W. anomalus produced more free amino acids and aromatic compounds, and there might be synergistic effects between these two strains. More characteristic soy sauce flavour compounds, such as benzaldehyde, HEMF, guaiacol and methyl maltol were detected in the sequentially inoculated moromi, and this sample showed higher scores in savoury, roasted and caramel intensities. These results confirmed that sequential inoculation of T. halophilus and W. anomalus could be a choice for the future production of moromi with good flavour and quality under a lower temperature.

Untargeted metabolomic profiling of Aspergillus sojae 3.495 and Aspergillus oryzae 3.042 fermented soy sauce koji and effect on moromi fermentation flavor

Aspergillus sojae 3.495 and Aspergillus oryzae 3.042 are essential microbial starters in fermented foods due to their high hydrolytic enzyme activities. However, their metabolic characteristics and differences during soy sauce fermentation are still unclear. This study identified 42 non-volatile differential metabolites based on UHPLC-MS/MS, among which 10 amino acids and derivatives and 4 non-volatile acids were high in A. sojae 3.495 koji, while 3 sugar alcohols, 5 flavonoids, and 4 fatty acids were prominent in A. oryzae 3.042. Further analysis revealed that higher acidic protease (1887.43 U/g) in A. sojae 3.495 and higher glucoamylase (2413.36 U/g) and cellulase (890.78 U/g) in A. oryzae 3.042 dominated the formation of differential metabolites. Metabolic pathway analysis showed that shikimate and phenylalanine metabolic pathways in A. sojae 3.495 were more active and enhanced 3-methyl-butanal and 2-phenylethanal in moromi fermentation. The more prominent pathways of sugar alcohol metabolism and flavonoid biosynthesis in A. oryzae 3.042 koji enhanced 2-methyl-butanoic acid and ethyl 3-methylbutyrate in moromi and significantly elevated reducing sugars (1.56 g/100 mL). Overall, comparative metabolomics elucidated the metabolic framework and flavor formation mechanisms of different inocula in soy sauce koji, providing ideas for scientific control of soy sauce fermentation and multi-strain koji-making for flavor enhancement.

Monitoring the growth dynamics of Tetragenococcus halophilus strains in lupine moromi fermentation using a multiplex-PCR system

ObjectiveThe microbiota of a seasoning sauce fermentation process is usually complex and includes multiple species and even various strains of one species. Moreover, composition and cell numbers of individual strains vary over the course of the entire fermentation. This study demonstrates the applicability of a multiplex PCR system to monitor growth dynamics of Tetragenococcus (T.) halophilus strains in order to evaluate their performance and help to select the most competitive starter strain.ResultsIn a previous study we isolated T. halophilus strains from multiple lupine moromi fermentation processes and characterized them. In this study we wanted to monitor the growth dynamics of these strains in a competitive lupine moromi model fermentation process using a multiplex PCR system. Therefore, pasteurized lupine koji was inoculated with eight different T. halophilus strains, six from lupine moromi, one from an experimental buckwheat moromi fermentation process and the type strain DSM 20,339T, to create the inoculated lupine moromi pilot scale fermentation process. With the multiplex PCR system, we could detect that all strains could grow in lupine moromi but, that TMW 2.2254 and TMW 2.2264 outperformed all other strains. Both strains dominated the fermentation after three weeks with cell counts between 4 × 106 to 4 × 107 CFU/mL for TMW 2.2254 and 1 × 107 to 5 × 107 CFU/mL for TMW 2.2264. The pH dropped to value below 5 within the first 7 days, the selection of these strains might be related to their acid tolerance.

Starter cultures inoculation procedure changes microbial community structure during low-salt moromi fermentation

Soy sauce is a condiment made of soybeans fermented in high salt concentration (18-22% NaCl) called moromi. Efforts to prevent sodium overconsumption has led to salt reduction in moromi. However, it could alter the microbial community dynamics during fermentation, resulting in undesirable sensory changes. This study investigates the effect of moromi starter cultures, Tetragenococcus sp. and Zygosaccharomyces rouxii, on microbial population changes during reduced-salt moromi (6% NaCl) fermentation. Both microbes were inoculated at different times, concentrations, and ratios, and the changes in total bacterial and yeast population during fermentation were monitored by culture on tryptic soy agar (TSA) and potato dextrose agar (PDA), respectively. The results showed that the inoculum size could shorten the bacterial exponential growth as it increased. Moreover, increasing the proportion of Tetragenococcus or Z. rouxii by two-fold could enhance the yeast and bacterial population, respectively by ∼3 log CFU/mL. Simultaneous inoculation suppressed bacterial growth, while sequential inoculation of Z. rouxii could maximize bacterial growth up to 7.38 log CFU/mL. However, a synergistic growth in simultaneous inoculation was observed when 5% inoculum with an equal ratio was used. This study could help researchers and manufacturers determine appropriate inoculation procedures to control fermentation better.

Amylase, protease, and lipase-producing microbes of local origin as potential starter cultures for low-salt moromi fermentation

High salt concentration (18%-22% NaCl) is essential for halophilic microbes to grow and develop aroma during soy sauce fermentation. Due to health concerns, recent efforts have focused on reducing salt during fermentation by introducing novel yeast species. However, the natural fermentation of soy sauce involves a more complex microbial community essential for achieving optimal sensory qualities. This study aimed to select potential bacterial and fungal species from traditional soy sauce factories in Indonesia for use in low-salt fermentation. Isolates that showed positive enzymatic activities at 20%, as well as 5% NaCl, were subjected to growth profile characterisation in tryptic soy broth (TSB) with different salt concentrations (0%, 5%, and 20%) and pH (4.0, 5.0, and 6.0). A total of 47 bacterial and 39 fungal isolates were obtained. Fourteen bacterial and 12 fungal isolates demonstrated positive enzymatic activities at 20% and 5% NaCl. The majority of bacterial strains were identified as Bacillus subtilis, while most of the fungal isolates belonged to the Aspergillus genus (Aspergillus oryzae and Aspergillus tamarii). The majority of Bacillus isolates performed rapid growth in 5% NaCl and relatively low pH (5.0), indicating their potential application in low-salt soy sauce production.

Microbial consortia and up-to-date technologies in global soy sauce production: A review

Soy sauce is an Oriental fermented condiment, and key ingredient in many Asian cuisines. As consumers around the world are becoming more adventurous with their eating choices and preferences, the demand for and popularity of Asian cuisines are increasing globally. The underlying basis of soy sauce fermentation is intricate microbial interactions which play a vital role in defining the quality, flavour, and smell of the resulting soy sauce. Traditional soy sauce fermentation consists of a two-step process: koji and moromi fermentation. Despite the presence of beneficial microorganisms in soy sauce, various harmful microorganisms can also be found during the koji or moromi step, thus resulting in soy sauce contamination. Therefore, studying the biodiversity and interactions of microorganisms is critical in ensuring soy sauce quality. The present review thus discusses in depth the various bacterial and fungal species that are either beneficial or harmful to soy sauce fermentation. The present review also discusses the advances in soy sauce fermentation such as the enhancement of gamma-aminobutyric acid (GABA) in soy sauce by microorganisms, the enhancement of soy sauce flavour by mixed starter culture, and by genome shuffling starter culture.

Correlation between bacterial diversity and flavor substances in Longgang soy sauce.

Longgang soy sauce is one of the traditional fermented condiments in China, but its bacterial community succession and its unique flavor development during the fermentation process are not well-investigated. This study evaluated the bacterial diversity, flavor changes, and their correlation during the fermentation of Longgang soy sauce. The results showed that Weissella was the dominant bacterial genus in the fermentation stage of sauce fermented grains. In the first 31 days of the moromi fermentation stage, a variety of bacterial genera such as Weissella, Halomonas, Bacteroides, Pseudomonas, and Tetragenococcus were the dominant bacteria. Our results showed that these bacteria have a significantly positive correlation with phenylethyl alcohol, ethyl acetate, and 3-methyl-1-butanol. As the fermentation progressed, a flora structure with Halomonas as the main bacterial genus was formed. This genus exhibited a significantly positive and positive correlation with 1-octanol, ethyl palmitate, heptanol, and 2-nonanol, which are the unique flavor components of Longgang soy sauce.

Insights into volatile flavor compound variations and characteristic fingerprints in Longpai soy sauce moromi fermentation via HS-GC-IMS and HS-SPME-GC× GC-ToF-MS

The variations in flavor substances across the different stages of Longpai soy sauce moromi fermentation were analyzed by HS-GC-IMS combined GC × GC-ToF-MS. A total of 374 VOCs were detected from soy sauce, and the highest content of VOCs were alcohols and esters, respectively. In the early stage fermentation, alcohols and aldehydes were the main flavor compounds. The content of esters, pyrazines, furans and S-containing compounds improved with continuous fermenting process. Based on VOCs, the samples could be delineated by clustering and PCA analysis. Of these, 63 VOCs were regarded as the marker VOCs of soy sauce in different fermentation stage. Alcohol, 1-butanol and 3-methyl-2-butenal provided fruity and mellow aroma at the early stage. Caramel-like, malty and sweet aroma could be enhanced by 3-methyl-2(5H)-furanone, 3-methylbutanol, methional and acetyl valeryl in the middle stage. Furthermore, furfuryl alcohol, 2(5H)-furanone, methyl cyclopentenolone, 2-Ethyl-6-methylpyrazine could be served as marker VOCs of matured Longpai soy sauce.

Genome Sequence of the Diploid Yeast Debaryomyces hansenii TMW 3.1188.

Debaryomyces hansenii TMW 3.1188 is a halotolerant diploid yeast that was isolated from lupine moromi fermentation. Here, we report on the 24.77-Mbp genome of a diploid strain of the species D. hansenii.

Chromohalobacter moromii sp. nov., a moderately halophilic bacterium isolated from lupine-based moromi fermentation

Three moderately halophilic strains, TMW 2.2308T, TMW 2.2299 and TMW 2.2304, were isolated from a lupine-based moromi fermentation. Initial identification based on their low molecular sub-proteome using mass spectrometry showed relation to the genus Halomonas, however, low score values indicated novelty. The comparison of 16S rRNA gene sequences placed these strains within the genus Chromohalobacter with C. japonicus CECT 7219T (99.67% 16S rRNA sequence similarity to strain TMW2.2308T), C. canadensis DSM 6769T (99.54%) and C. beijerinckii LMG 2148T (99.32%) being their closest relatives. However, average nucleotide highest identity values of TMW 2.2308T to C. beijerinckii LMG 2148T of 93.12% and 92.88% to C. japonicus CECT 7219T demonstrate that it represents a novel species within the genus Chromohalobacter with additional strains TMW 2.2299 (96.91%) and TMW 2.2304 (96.98%). The isolated strains were non-spore-forming, motile and able to grow at temperatures from 5 to 45 °C with an optimum at 37 °C. Growth of TMW 2.2308T occurs at 5 to 25% (w/v) NaCl with optimum growth between 10and 12.5%. The genome of TMW 2.2308T has a size of 3.47 Mb and a G + C content of 61.0 mol%. The polyphasic evidence lead to the classification of TMW 2.2308T, TMW 2.2299 and TMW 2.2304 as members of a novel species of the genus Chromohalobacter. We propose a novel species as Chromohalobacter moromii sp. nov., with TMW 2.2308T (=DSM113153T =CECT30422T) as the type strain.

Effects of Tartary buckwheat on physicochemical properties and microbial community of low salt natural fermented soybean paste

It is a valuable attempt to use Tartary buckwheat as the raw material of soybean paste due to its functional composition. In this research, the soybean pastes were prepared with Tartary buckwheat flour and wheat flour by low salt natural fermentation (10%, w/v NaCl), respectively; the physicochemical properties and microbial community structure of samples prepared from the two groups were investigated. The results showed that Tartary buckwheat promoted the accumulation of protease, total acid (TA), amino acid nitrogen (AAN) significantly compared with the control group during fermentation (p < 0.05). Both groups had similar bacterial communities, Klebsiella, unidentified_Chloroplast, Bacillus, Staphylococcus, Enterococcus, Acinetobacter, Weissella were present in all samples. But the fungal communities of the two groups were significantly different. The dominant fungi in all samples were Rhizopus and Aspergillus. In moromi fermentation, the Issatchenkia presented only in buckwheat group, Stenocarpella and Gibberella were characteristic fungi of wheat flour group. The changes of biodiversity and community structure showed that the microbial communities of the two groups experienced different succession patterns. The results can provide a reference for the further development of low salt Tartary buckwheat soybean paste and the study of microbial community succession in soybean paste fermentation.

Effect of Lactobacillus plantarum or Enterococcus faecalis as co-inoculants with Aspergillus oryzae in koji making on the physicochemical properties of soy sauce.

Soy sauce fermentation is largely mediated by bacteria, including several non-salt-tolerant bacterial species. The present study aimed to compare the use of Aspergillus oryzae alone or in co-inoculation with Lactobacillus plantarum or Enterococcus faecalis on the resulting enzymatic activity, content of phenolic compounds, free amino acids, organic acids, and antioxidant capacity of soy sauce. Co-inoculation significantly increased acid protease, β-glucosidase, and pectinase activities during koji making. Additionally, the contents of total phenols, total flavonoids, organic acids, amino acids, isoflavones, as well as the antioxidant capacity were higher during moromi fermentation using co-inoculants compared to those obtained when A. oryzae was used as single inoculant. Overall, co-inoculation of A. oryzae with L. plantarum or E. faecalis during koji making was shown to effectively promote the production of active substances in soy sauce. PRACTICAL APPLICATION: In this study, Aspergillus oryzae in mixed inoculation with Lactobacillus plantarum or Enterococcus faecalis could be effectively used in koji making for soy sauce. The use of co-inoculants in koji making effectively promoted the production of active substances in soy sauce, hence it is improving health benefits, whilst diversifying the use of raw materials and the application value of soybean meal.

Optimization of moromi fermentation parameters to nutritional content of oyster mushrooms sauce (Pleurotus spp.) by using response surface methodology

Oyster mushrooms are used as raw materials for sauce because of their enhanced nutritious and sensory flavour. The study was aimed to select the optimal parameters of moromi fermentation of oyster mushrooms sauce. Optimizing moromi fermentation from oyster mushrooms by response surface method (RSM) was conducted with 3 factors including salt concentration (15-25% w/v), the brine ratio [150-250% (compared to raw materials, v/w)] and fermentation time (50-70 days). The central composite design used for optimizing the effect of salt concentration, amount of brine and fermentation time showed that the parameters were in agreement with the quadratic regression models well with R2 &gt;0.95. Based on the response surface plots, the optimal parameters of moromi fermentation were salt concentration 20% (w/v), brine ratio 190% (compared to raw materials, v/w) and 60 days of fermentation time. In the optimized condition, the chemical composition (reducing sugar, amino protein, total acids) and the bioactive compounds (βglucan, total phenolic, total flavonoid) (per kg of dry matter) of fermentation sauce were 258.94 g, 204.76 g, 99.06 g, 86.57 g, 108.32 mg TAE and 93.78 mg QE, respectively

A new sight on soy isoflavones during the whole soy sauce fermentation process by UPLC-MS/MS

Soy sauce fermentation process includes koji-making and moromi fermentation stages, with the moromi stage consisting of solid (okara) and liquid (raw soy sauce) phases. Evolution of soy isoflavones in koji, okara and raw soy sauce samples was analyzed systematically, and the rule regarding the formation of soy isoflavones during the whole fermentation process was firstly figured out. Fifteen kinds of soy isoflavones were detected by UPLC-MS/MS. Following the fermentation process, the soy isoflavones profiles changed dramatically in the initial moromi stage (1 d-15 d). The concentrations of glucosides, malonylglucosides and acetylglucosides decreased and disappeared around at okara-30 d, while the concentrations of aglycones and hydroxyl aglycones increased and attained the peak at 90 d of the moromi stage. Additionally, antioxidant capacities of samples were positively correlated with the concentrations of aglycones and hydroxyl aglycones. Interestingly, in each sample, the antioxidant contribution value of hydroxydaidzein was around 100–500 times higher than that of daidzein, which demonstrated that aglycones, especially hydroxyl aglycones, were the key isoflavones contributed to the antioxidant properties of soy sauce.

Microbiota dynamics and volatile compounds in lupine based Moromi fermented at different salt concentrations

Fermented soy sauces are used as food seasonings in Eastern countries and all over the world. Depending on their cultural origins, their production differs in parameters such as wheat addition, temperature, and salt concentration. The fermentation of lupine seeds presents an alternative to the use of soybeans; however, the microbiota and influencing factors are currently unknown. In this study, we analyse the microbiota of lupine Moromi (mash) fermentations for a period of six months and determine the influence of different salt concentrations on the microbiota dynamics and the volatile compound composition. Cultured microorganisms were identified by protein profiling using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 16S rRNA gene amplicon sequencing provided an overview of the microbiota including non-cultured bacteria. The volatile compounds were determined by gas chromatography–mass spectrometry (GC–MS). At all salt concentrations, we found that Tetragenococcus halophilus (up to 1.4 × 109 colony forming units (CFU)/mL on day 21) and Chromohalobacter japonicus (1.9 × 109 CFU/mL, day 28) were the dominating bacteria during Moromi fermentation. Debaryomyces hansenii (3.6 × 108 CFU/mL, day 42) and Candida guilliermondii (2.2 × 108 CFU/mL, day 2) were found to be the most prevalent yeast species. Interestingly, Zygosaccharomyces rouxii and other yeasts described as typical for soy Moromi were not found. With increasing salinity, we found lower diversity in the microbiota, the prevalence-gain of typical species was delayed, and ratios differed depending on their halo- or acid tolerance. GC–MS analysis revealed aroma-active compounds, such as pyrazines, acids, and some furanones, which were mostly different from the aroma compounds found in soy sauce. The absence of wheat may have caused a change in yeast microbiota, and the use of lupine seeds may have led to the differing aromatic composition. Salt reduction resulted in a more complex microbiome, higher cell counts, and did not show any spoiling organisms. With these findings, we show that seasoning sauce that uses lupine seeds as the sole substrate is a suitable gluten-free, soy-free and salt reduced alternative to common soy sauces with a unique flavour.

Screening and Application of Bacillus spp. Promoting Moromi Fermentation

Screening and Application of Bacillus spp. Promoting Moromi Fermentation