Recurrent Bacillus subtilis Var. Natto Bacteremia and Review of the Literature on Bacillus subtilis: The First Case Report.

BACKGROUND Fermented foods, such as yogurt, are often considered healthy; however, there have been numerous reported cases of bacteremia associated with their consumption. In this report, we present a case of Bacillus subtilis var. natto (B. subtilis var. natto) bacteremia related to the consumption of natto, a traditional Japanese food made from fermented soybeans. We also conducted a literature review on B. subtilis bacteremia. CASE REPORT We report the case of a 41-year-old woman who presented with fever, had a medical history of congenital liver fibrosis, and experienced recurrent B. subtilis var. natto bacteremia along with acute cholangitis. Although she discontinued eating natto, she developed pyogenic thrombophlebitis due to B. subtilis var. natto. We successfully treated her with meropenem and an anti-coagulant. To investigate the management and prognosis of B. subtilis var. natto bacteremia, we conducted a literature review of B. subtilis intra-abdominal infection. We identified 17 papers describing 30 cases of B. subtilis intra-abdominal infection, 4 cases of which were caused by B. subtilis var. natto; the median age of the patients was 71 years (range, 15-96 years), 14 patients (47%) were female, and 3 patients (10%) died. From our findings, our case was the only one of recurrent B. subtilis var. natto infection. Even after patients discontinue eating natto, they should be carefully monitored. CONCLUSIONS Due to advancements in PCR identification techniques, case reports of infections caused by B. subtilis var. natto are increasing.

Unveiling the aromatic differences of low-salt Chinese horse bean-chili-paste using metabolomics and sensomics approaches.

To achieve salt reduction while ensuring flavor quality of Chinese horse bean-chili-paste (CHCP), we comprehensively explored the effect of indigenous strains Tetragenococcus halophilus and Candida versatilis on the aroma profiles of low-salt CHCP by metabolomics and sensomics analysis. A total of 129 volatiles and 34 aroma compounds were identified by GC × GC-MS and GC-O-MS, among which 29 and 20 volatiles were identified as significant difference compounds and aroma-active compounds, respectively. Inoculation with the two indigenous strains could effectively relieve the undesired acidic and irritative flavor brought by acetic acid and some aldehydes in salt-reduction samples. Meanwhile, inoculated fermentation provided more complex and richer volatiles in low-salt batches, especially for the accumulation of 3-methylbutanol, 1-octen-3-ol, benzeneacetaldehyde, phenylethyl alcohol, and 4-ethyl-phenol etc., which were confirmed as essential aroma compounds of CHCP by recombination and omission tests. The research elucidated the feasibility of bioturbation strategy to achieve salt-reducing fermentation of fermented foods.

Impacts of Aspergillus oryzae 3.042 on the flavor formation pathway in Cantonese soy sauce koji.

To investigate the mechanism of flavor formation during the traditional preparation Cantonese soy sauce koji (TP), the changes of microorganisms, physicochemical properties, and flavor compounds in TP were comprehensively and dynamically monitored by absolute quantitative methods. Results demonstrated that inoculating Aspergillus oryzae 3.042 in TP was crucial role in enhancing enzyme activity properties. Absolute quantification of flavor combined with multivariate statistical analysis yielded 5 organic acids, 15 amino acids, and 2 volatiles as significantly different flavors of TP. Amplicon sequencing and RT-qPCR revealed that the dominant genera were Staphylococcus, Weissella, Enterobacter, Lactic streptococci, Lactobacillus, and Aspergillus, which exhibited a increasing trend in TP. Correlation analysis exhibited that Staphylococcus and Aspergillus were the pivotal genera contributing to the enzyme activities and flavor of TP. The flavor formation network involved lipid and protein degradation, carbohydrate metabolism and other pathways. Simultaneously, TP can appropriately increase the fermentation time to improve product quality.

Characterization of key aroma compounds in soy sauce flavor baijiu by molecular sensory science combined with aroma active compounds reverse verification method.

The distinctive flavor profile of soy sauce flavor baijiu (SAB) is shaped by its unique aroma compounds. The characteristic aroma compounds in Langjiu soy sauce flavor baijiu (LSAB) were explored based on molecular sensory science. A total of 66 aroma active compounds were identified by gas chromatography-olfactometry (GC-O) combined with aroma extract dilution analysis (AEDA), and 6 important unknown sulfur compounds were identified using the aroma active compounds reverse verification method (ACRVW). A total of 39 key aroma compounds were determined to have odor activity values (OAVs) ≥ 1. The aroma contribution of aroma components was verified by aroma recombination and aroma omission experiments. 15 characteristic aroma compounds were identified in LSAB. Meanwhile, a simple and easy-to-understand sensory expression language was described to fully understand the style characteristics of LSAB. Overall, the present paper offers insights into research uncovering the key "sauce flavor" of soy sauce flavor baijiu.

Decoding temperature-driven microbial community changes and flavor regulation mechanism during winter fermentation of soy sauce.

The flavor regulation of soy sauce fermented in winter is imminent challenge for the industry, while fermentation temperature is considered as an effective method to fortify soy sauce flavor. Thus, industrial-level fermentation systems with controlled temperature at 30°C (SSCT) and regular temperature (SSRT) in winter were designed to elucidate molecular basis and microbial regulatory mechanism of temperature-controlled flavor enhancement of soy sauce. Sensory evaluation suggested 30°C fermentation enhanced caramel-like, floral, fruity, roasted nut and smoky aroma. A total of 160 volatiles were identified, of which 39 components were evaluated for odor activity value (OAV). Eleven volatiles were determined as the odor markers distinguishing the aroma profiles of SSRT and SSCT, among which 2,5-dimethyl-4-hydroxy-3(2H)-furanone (HDMF, caramel-like), ß-damascenone (floral), ethyl 2-methylpropanoate (fruity), ethyl acetate (fruity) and 2/3-methyl-1-butanol (malty, alcoholic) were largely responsible for the flavor enhancement. Moreover, high-throughput sequencing results demonstrated the temperature intervention induced more differential bacterial structure (R = 0.324, P = 0.001) than fungal structure (R = 0.069, P = 0.058). Correlation analysis revealed dominant and low-abundance genus together drove the formation and variation of volatile profile, particularly Weissella, Tetragenococcus, Starmerella and Pediococcus. Representatively, the formation pathways of key aroma substances HDMF and 5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone (HEMF) were elaborated. Both temperature-mediated abiotic reactions and gene functions of microbiota were proposed to favor the yields of HDMF and C5 precursor of HEMF, whereas the small populations of Zygosaccharomyces and insufficient acetaldehyde limited the elevation of the HEMF level through the biosynthesis pathway. This study provided the practical and theoretical basis for the industrial applications of temperature control in soy sauce fermentation.

High-intensity ultrasound promoted the maturation of high-salt liquid-state soy sauce: A mean of enhancing quality attributes and sensory properties.

High-intensity ultrasound was used as a means to promote maturation of soy sauce. The optimal conditions for ultrasound treatment were 90℃ at an ultrasound intensity of 39.48 W/cm2 for 60 min. The total reducing sugars and soluble salt-free solids content was significantly increased after ultrasound-assisted maturation. The free amino acid content was significantly decreased, mainly due to the Maillard reaction (MR). The promoted MR produced several types of flavor compounds, including esters, pyrazines, and ketones, which imparted an attractive aroma to the maturated soy sauce. The proportion of peptides with a molecular weight of 1-5 kDa provided umami as an important flavor characteristic, and the content in the ultrasound-matured soy sauce (10.19 %) was significantly higher than that in the freshly prepared soy sauce (8.34 %) and the thermally treated sample (8.89 %). Ultrasound-assisted maturation would improve product quality and meanwhile, shorten the duration and reduce the cost for the soy sauce industry.

Untargeted metabolomics revealed the effect of soybean metabolites on poly(γ-glutamic acid) production in fermented natto and its metabolic pathway.

BACKGROUND: Natto mucus is mainly composed of poly(γ-glutamic acid) (γ-PGA), which affects the sensory quality of natto and has some effective functional activities. The soybean metabolites that cause different γ-PGA contents in different fermented natto are unclear. RESULTS: In this study, we use untargeted metabolomics to analyze the metabolites of high-production γ-PGA natto and low-production γ-PGA natto and their fermented substrate soybean. A total of 257 main significantly different metabolites with the same trend among the three comparison groups were screened, of which 114 were downregulated and 143 were upregulated. Through the enrichment of metabolic pathways, the metabolic pathways with significant differences were purine metabolism, nucleotide metabolism, fructose and mannose metabolism, anthocyanin biosynthesis, isoflavonoid biosynthesis and the pentose phosphate pathway. CONCLUSION: For 114 downregulated main significantly different metabolites with the same trend among the three comparison groups, Bacillus subtilis (natto) may directly decompose them to synthesize γ-PGA. Adding downregulated substances before fermentation or cultivating soybean varieties with the goal of high production of such substances has a great effect on the production of γ-PGA by natto fermentation. The enrichment analysis results showed the main pathways affecting the production of γ-PGA by Bacillus subtilis (natto) using soybean metabolites, which provides a theoretical basis for the production of γ-PGA by soybean and promotes the diversification of natto products. © 2023 Society of Chemical Industry.

Evaluation of the differences between low-salt solid-state fermented soy sauce and high-salt diluted-state fermented soy sauce in China: from taste-active compounds and aroma-active compounds to sensory characteristics.

BACKGROUND: The present study aimed to determine the components related to sensory properties in soy sauce and to characterize the differences between low-salt solid-state fermented soy sauce (LSFSS) and high-salt diluted-state fermented soy sauce (HDFSS). The taste and aroma active components of 18 commercially available soy sauces (eight types of LSFSS and 10 types of HDFSS) were characterized. The relationship between these compounds, soy sauce samples, and sensory properties was modeled by partial least squares regression. RESULTS: The analysis showed that the 11 taste-active components, including glutamic acid, glycine, alanine, threonine, malic acid, citric acid, tartaric acid, acetic acid, lactic acid, reducing sugar and salt, contributed greatly to the taste of soy sauce. In addition, umami, saltiness and sweetness are the characteristic tastes of HDFSS, whereas sourness and bitterness were the characteristic tastes of LSFSS. At the same time, seven aroma-active compounds, namely 4-ethyl-2-methoxyphenol, ethanol, 3-methyl-1-butanol, ethyl acetate, 2-phenethyl alcohol, 3-methyl thiopropanol and 2-ethyl-4-hydroxy-5-methylfuran-3-one, played a decisive role in the flavor of soy sauce. In addition, HDFSS presented the aroma attributes of smoky, alcoholic, floral, fruity and caramel-like, whereas LSFSS mainly presented sour and malty aroma attributes. CONCLUSION: The present study reveals new insight into the relationship between the chemical composition and sensory characteristics of soy sauce, which is of great significance for developing an objective measurement system and providing a theoretical basis to improve the sensory quality of soy sauce. © 2023 Society of Chemical Industry.

In vitro protein digestibility of different soy-based products: effects of microstructure, physico-chemical properties and protein aggregation.

This study investigates the effects of protein structure and food microstructure on the in vitro protein gastrointestinal digestibility of different soy-based products, such as soy drink, reconstituted soy drink powder, firm tofu, and yuba. The results of the chemical cross-linking analysis showed that hydrogen bonds and hydrophobic interactions were the main forces driving protein aggregation in (reconstituted) soy drink powder and firm tofu, whereas disulphide bonds were significantly more important for soy drink and yuba. The ß-sheet content of soy drink (36.5%) was lower than that of yuba (43.3%), but significantly higher than those of soy drink powder (23.2%) and firm tofu (29.8%). The in vitro protein digestibility decreased in the order of firm tofu > reconstituted soy drink powder > yuba > soy drink. Principal component analysis showed that protein gastrointestinal digestibility was positively correlated with the surface SH content and soluble protein content released by SDS + urea (SB-SA) but negatively correlated with the disulphide bonds and ß-sheet content for the four soybean products.

Rapid Sample Enrichment, Novel Derivatization, and High Sensitivity for Determination of 3-Chloropropane-1,2-diol in Soy Sauce via High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

A novel, simplified derivatization method and a rapid sample preparation process using carbon yarn as a sorbent for the determination of 3-chloropropane-1,2-diol (3-MCPD) in soy sauce via high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. 3-MCPD was first enriched and purified with carbon yarn and then eluted with a methanol-water solution. Subsequently, the analyte underwent derivatization with p-(dimethylamino)-phenol for sensitive detection via HPLC-MS/MS. The limit of detection and the limit of quantitation for 3-MCPD were validated to be 0.5 and 1.0 µg/kg, respectively. Spiking experiments showed recoveries between 83 and 94%, with a relative standard deviation of ≤10%. The method was further validated with a certified reference material. Furthermore, 11 real soy sauce samples from local markets were tested by using this method. These results reveal the widespread 3-MCPD contamination. Consequently, this study offers a preferable alternative for the sensitive, accurate, and precise determination of 3-MCPD in soy sauce.

Exploring the core functional microbiota related with flavor compounds in fermented soy sauce from different sources.

Flavor, the most important quality index of soy sauce, is mostly influenced by the microbiota in fermented food ecosystem, however, the association between microorganisms and soy sauce flavor is still poorly understood. Therefore, the bacterial and fungal profiles, physicochemical parameters, and flavor compounds (9 organic acids, 17 free amino acids and 97 volatile flavor compounds) of 5 different source soy sauce were investigated using high-throughput sequencing, HPLC, amino acid analyzer and SPME/LLE-GC-MS, and their correlations were explored. A total of 3 fungal genera and 12 bacterial genera were identified as potential flavor-producing microorganisms by multivariate data and correlation analysis. Notably, Lactobacillus and Tetragenococcus were strongly positively correlated with succinic acid and lactic acid, respectively. Moreover, not only fungi, but also bacteria were found to be closely correlated with volatiles. Finally, 5 screened potential flavor-producing microorganisms were validated using a rapid fermentation model, with multiple strains showing the potential to improve the soy sauce flavor, with Lactobacillus fermentum being the most significant. Our research will provide a theoretical basis for the regulation and enhancement of soy sauce flavor.

Fractionation, identification and umami characteristics of flavor peptides in natural brewed soy sauce.

To investigate the umami mechanisms and characteristics of soy sauce flavor peptides, four fractions from natural brewed soy sauce were separated using ultrafiltration and Sephadex G-15 gel filtration chromatography. Sensory and ligand-receptor interaction tests showed that the umami strengths of the fractions were related as follows: U1 > U2, G3 > G2, and G3 > U1. Peptide identification revealed that the < 550-Da peptides might be the major contributors to the umami taste of U1 and G3. The higher umami strength of G3 might be attributable to its higher content of umami peptides. G3's concentration-relative umami intensity curve was plotted using a two-alternative forced choice test. It was also revealed that less sourness, higher saltiness and cool (4 ℃) and hot (50 ℃) serving conditions were conductive to the umami perception of G3. The results could provide a reference for the application of soy-sauce flavor peptides in food.

Upcycling of Soy Whey with Ischnoderma benzoinum toward Production of Bioflavors and Mycoprotein.

Soy whey is becoming a worldwide issue as a liquid waste stream that is often discarded after tofu, soy protein, and soy-based dairy alternative manufacturing. This study established a model to produce a bioflavor and mycoprotein using fermentation of soy whey by a basidiomycete Ischnoderma benzoinum. Under the dedicated control of a fermentation system, an intense almond-like and sweetish aroma was perceived by a trained sensory panel (n = 10) after fermentation of pure soy whey within 20 h. By application of direct immersion-stir bar sorptive extraction combined with gas chromatography-mass spectrometry-olfactometry (DI-SBSE-GC-MS-O), around 1.0 mg/L benzaldehyde and 1.1 mg/L 4-methoxybenzaldehyde imparting a pleasant almond-like odor note were determined in the fermented soy whey with I. benzoinum. Concurrently, a certain of amount of the dry mass of I. benzoinum was accumulated with 73.2 mg/g crude protein and seven essential amino acids.

Characterization of the key aroma compounds in soy sauce by gas chromatography-mass spectrometry-olfactometry, headspace-gas chromatography-ion mobility spectrometry, odor activity value, and aroma recombination and omission analysis.

Most previous studies on volatile compounds in soy sauce were performed by gas chromatography-mass spectrometry (GC-MS). In this study, the volatile compounds of high-salt liquid-state fermentation soy sauce (HLFSS) were analyzed qualitatively and quantitatively by GC-MS and headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). One hundred and seventy-four substances were detected using the two instruments, 87 by HS-GC-IMS and 127 by GC-MS. Aldehydes (26), ketones (28), esters (29), and alcohols (26) were the main compounds in HLFSS. In addition, ethyl pyruvate, (E)-2-pentenal and diethyl propanedioate were detected by HS-GC-IMS, which were previously not detected in HLFSS. Forty-eight aromatics including 34 key ones were identified by gas chromatography-olfactometry. Phenylacetaldehyde, methional, 2-methylbutanal, 1-octen-3-ol, ethyl acetate, 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone, 4-hydroxy-2,5-dimethyl-3(2H)-furanone and 4-ethyl guaiacol were identified as the main aroma compounds in HLFSS by aroma recombination and omission test. This study laid foundation for developing flavor assessment standards for soy sauce.

A systematic review on the flavor of soy-based fermented foods: Core fermentation microbiome, multisensory flavor substances, key enzymes, and metabolic pathways.

The characteristic flavor of fermented foods has an important impact on the purchasing decisions of consumers, and its production mechanisms are a concern for scientists worldwide. The perception of food flavor is a complex process involving olfaction, taste, vision, and oral touch, with various senses contributing to specific properties of the flavor. Soy-based fermented products are popular because of their unique flavors, especially in Asian countries, where they occupy an important place in the dietary structure. Microorganisms, known as the souls of fermented foods, can influence the sensory properties of soy-based fermented foods through various metabolic pathways, and are closely related to the formation of multisensory properties. Therefore, this review systematically summarizes the core microbiome and its interactions that play an active role in representative soy-based fermented foods, such as fermented soymilk, soy sauce, soybean paste, sufu, and douchi. The mechanism of action of the core microbial community on multisensory flavor quality is revealed here. Revealing the fermentation core microbiome and related enzymes provides important guidance for the development of flavor-enhancement strategies and related genetically engineered bacteria.

Measurements of elemental iodine in soy sauces in Taiwan using a modified microplate method.

Background: Soy sauce is widely used in a variety of Asian dishes to enhance flavor. Soybean and most soybean products, including soy sauces, are listed as prohibited foods in a low iodine diet. However, the iodine content in soy sauces is largely unknown. The aim of this study was to determine the iodine content in domestic soy sauces in Taiwan. Methods: Twenty-five different kinds of soy sauces were diluted with distilled water and with a dilution factor of fifty or above. Iodine concentrations of the diluted samples were measured colourimetrically based on the Sandell-Kolthoff reaction by a modified microplate method. All the measurements were repeated twelve times on three different days for determination of mean and standard deviation (SD), and coefficients of variance (CV). Serial dilution and recovery tests were also performed for validation. The results were confirmed by an inductively coupled plasma mass spectrometry (ICP-MS) method. Results: Among the twenty-five surveyed soy sauces, most of them (n=22) were iodine-free (<16 ug/L, and thus un-detectable). The iodine concentrations (mean ± SD) of the three iodine-containing soy sauces were 2.7 ± 0.1, 5.1 ± 0.2, and 10.8 ± 0.6 mg/L, respectively. The inter-assay, intra-assay and total CVs were all <5.3% for the modified microplate method. The results obtained by ICP-MS were consistent with those of the modified microplate method. The recovery rates in the serial dilution test and recovery test ranged from 94.7% to 118.6%. Two of the three iodine-containing soy sauces were supplemented with kelp extract, while the other one without kelp extract had the highest amount of salt among the three iodine-containing soy sauces. Therefore, we postulate that iodized salt instead of kelp extract is the source of higher iodine content in that sauce. Conclusion: The results suggest that most soy sauces are iodine-free and may be allowed during low iodine diets.

Comprehensive investigation on volatile and non-volatile metabolites in low-salt doubanjiang with different fermentation methods.

Doubanjiang is a well-known fermented condiment in China, but the high-salt concentration in its traditional manufacture process greatly lengthens the fermentation time, and leads to potential health risks. Here, the effects of salt reduction and co-inoculated starters (Tetragenococcus halophilus and Zygosaccharomyces rouxii) on the volatile metabolites (VMs) and non-volatile metabolites (NVMs) of doubanjiang were investigated using metabolomics technology and chemometrics analysis. Results showed that 75 VMs were identified, and 12 of them had significant aroma contribution (ROVAs ≥ 1). In addition, 106 NVMs were defined as significantly different metabolites (p < 0.05; VIP ≥ 1). Salt reduction could significantly increase the concentrations of VMs, but this strategy also promoted some undesirable odors like 2-phetylfuran and hexanoic acid, which could be totally suppressed by inoculation of starter. Moreover, the two starters improved amino acid, ester, and acid metabolites. This study provides a deeper insight into the development of low-salt fermented foods.

Ratio of Histamine-Producing/Non-Histamine-Producing Subgroups of Tetragenococcus halophilus Determines the Histamine Accumulation during Spontaneous Fermentation of Soy Sauce.

Strain specificity (within-species variation) of microorganisms occurs widely in nature. It might affect microbiome construction and function in a complex microbial environment. Tetragenococcus halophilus, a halophilic bacterium that generally is used in high salt food fermentation, consists of two histamine-producing and non-histamine-producing subgroups. It is unclear whether and how the strain specificity of histamine-producing capacity influences the microbial community function during food fermentation. Here, based on systematic bioinformatic analysis, histamine production dynamic analysis, clone library construction analysis, and cultivation-based identification, we identified that T. halophilus is the focal histamine-producing microorganism during soy sauce fermentation. Furthermore, we discovered that a larger number and ratio of histamine-producing subgroups of T. halophilus significantly contributed more histamine production. We were able to artificially decrease the ratio of histamine-producing to non-histamine-producing subgroups of T. halophilus in complex soy sauce microbiota and realized the reduction of histamine by 34%. This study emphasizes the significance of strain specificity in regulating microbiome function. This study investigated how strain specificity influenced microbial community function and developed an efficient technique for histamine control. IMPORTANCE Inhibiting the production of microbiological hazards under the assumption of stable and high-quality fermentation is a critical and time-consuming task for the food fermentation industry. For spontaneously fermented food, it can be realized theoretically by finding and controlling the focal hazard-producing microorganism in complex microbiota. This work used histamine control in soy sauce as a model and developed a system-level approach to identify and regulate the focal hazard-producing microorganism. We discovered that the strain specificity of focal hazard-producing microorganisms had an important impact on hazard accumulation. Microorganisms frequently exhibit strain specificity. Strain specificity is receiving increasing interest since it determines not only microbial robustness but also microbial community assembly and microbiome function. This study creatively explored how the strain specificity of microorganisms influenced microbiome function. In addition, we believe that this work provides an excellent model for microbiological hazard control which can promote future work in other systems.

Microbiology, flavor formation, and bioactivity of fermented soybean curd (furu): A review.

Soybeans are an important plant-based food but its beany flavor and anti-nutritional factors limit its consumption. Fermentation is an effective way to improve its flavor and nutrition. Furu is a popular fermented soybean curd and mainly manufactured in Asia, which has been consumed for thousands of years as an appetizer because of its attractive flavors. This review first classifies furu products on the basis of various factors; then, the microorganisms involved in its fermentation and their various functions are discussed. The mechanisms for the formation of aroma and taste compounds during fermentation are also discussed; and the microbial metabolites and their bioactivities are analyzed. Finally, future prospects and challenges are introduced and further research is proposed. This information is needed to protect the regional characteristics of furu and to regulate its consistent quality. The current information suggests that more in vivo experiments and further clinical trials are needed to confirm its safety and the microbial community needs to be optimized and standardized for each type of furu to improve the production process.