Synthesis, taste characteristics and taste mechanism of N-lactoyl leucine from soy sauce using sensory analysis and UPLC-MS/MS.

Recently, amino acid derivatives gradually gained attention, but studies on N-lactoyl-leucine (Lac-Leu) and N-lactoyl-isoleucine (Lac-Ile) are limited. This study aims to explore the contributions of Lac-Leu and Lac-Ile to soy sauce. Lac-Leu and Lac-Ile were synthesized via enzymatic synthesis method catalyzed by Tgase. The mixed solutions containing Lac-Leu were found to have greater taste improvement than those containing Lac-Ile. Sensory evaluation indicated the sour, bitter, and astringent taste of Lac-Leu in water as well as its kokumi, astringent, and umami-enhancing taste in MSG solution. The taste threshold and umami-enhancing threshold of Lac-Leu measured by TDA and cTDA, respectively, were 0.08 mg/mL and 0.16 mg/mL. Molecular docking of Lac-Leu and Lac-Ile with the kokumi receptor CaSR and the umami receptors T1R1 and T1R3 indicated that Lac-Leu had higher affinities with receptors than Lac-Ile. These findings demonstrated the underlying contribution Lac-Leu made to soy sauce, indicating its potential to improve the flavor quality of soy sauce.

Tailored UPRE2 variants for dynamic gene regulation in yeast.

Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of HAC1 enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories.

Effect of fermentation containers on the taste characteristics and microbiota succession of soy sauce.

Modernization of the traditional fermentation industry has been a major trend recently, such as the upgrading of fermentation containers. This study investigated the taste differences and their material basis of soy sauce fermented in tank and pond (SSFT and SSFP), and further explore the key influencing factors of taste. The intensities of umami, kokumi and sour taste in SSFT were weaker than SSFP, which were associated with 9 basic taste-active compounds, including acetic acid, lactic acid, propanedioic acid, citric acid, glutamic acid, alanine, tyrosine, d-galactose and erythritol. Moreover, 270 peptides and amino acid derivatives were potential compounds for taste difference, of which 78 % were more abundant in SSFP. Five bacterial genera (Kocuria, Tetragenococcus, Pediococcus, Staphylococcus, Weissella) and 4 fungal genera (Wickerhamiella, Millerozyma, Candida, Zygosaccharomyces) may be the functional core microbe for flavor differences in SSFT and SSFP. This study will provide theoretical value for quality improvement in the modern large-scale production of soy sauce.

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.

Exploration of optimal preparation strategy of Chenpi (pericarps of Citrus reticulata Blanco) flavouring essence with great application potential in sugar and salt-reduced foods.

To obtain flavouring essence with application potential in sugar and salt-reduced foods, the optimal strategy for extraction and microencapsulation of essential oil (EO) from Chenpi was investigated. UPLC-QTOF-MS/MS and liquid-liquid-extraction-GC-MS confirmed the selectivity for volatiles ranked in hydrodistillation > supercritical fluid extraction > solvent extraction. The aroma characteristic of Chenpi EO was distinguished by 33 key volatiles (screened out via headspace-SPME-GC-MS) and quantitative descriptive analysis. EO extracted by supercritical fluid extraction was preferred for preserving the original aroma of Chenpi and displaying more fruity, honey and floral. Chenpi flavouring essence with superior encapsulation efficiency, particle size, water dispersibility, and thermostability was obtained through optimally microencapsulating EO with gum arabic and maltodextrin (1:1) by high-pressure homogenization coupled with spray drying. Chenpi flavouring essence was able to reduce the usage of sugar and salt by 20 % via enhancing flavour perception of sweetness and saltiness. This study first developed a flavouring essence promisingly effective in both sugar and salt-reduced foods.

Preparation and Kokumi Properties of N-Acetyl-Val/Leu/Ile/Met/Phe in the Presence of Acetic Acid and Amino Acid: A Commercially Available Transglutaminase and Protease A 2SD.

Kokumi is a beneficial feeling for the evaluation of food quality, and thus, preparing and understanding the taste properties of kokumi compounds are important for the flavor of food. N-acetyl-Val/Leu/Ile/Met/Phe/Trp/Tyr is a type of kokumi compound found in food and usually prepared by chemical reagents. In this study, we first prepared these six kokumi compounds using transglutaminase and protease A2SD in aqueous solution by using amino acids and acetic acid as substrates and evaluated their kokumi characteristics. HPLC and LC-MS were used to identify quantitative N-acetyl amino acids. Using Phe and acetic acid as substrates, transglutaminase and protease A2SD showed the highest yields for N-acetyl-Phe of 22.75 and 42.21%, respectively, under the optimal conditions. For N-acetyl-Val/Leu/Ile/Met/Trp/Tyr, these two enzymes showed the synthesis yield in the ranges of 2.22-20.12 and 0.75-12.91%, respectively. Six N-acetyl-amino acids were succesully enriched by ethyl acetate with a recovery over 50% and purity over 95%. Sensory evaluation found that N-acetyl-Val/Leu/Ile/Met/Phe are kokumi compounds that enhance sweet, umami, and salt tastes in 5% sucrose, 0.3% NaCl, and 0.5% sodium glutamate, especially N-acetyl-Val, with the salt- and umami-enhancing threshold values of 0.63 and 1.25 g/L, respectively. Therefore, transglutaminase and protease A2SD for the synthesis of partial N-acetyl amino acid might have the potential to be applied in food as a kokumi compound.

Enhancing Kokumi Sensation and Reducing Bitterness in Acid-Hydrolyzed Vegetable Proteins through Lactate and Thermal Processing.

Previous studies have demonstrated that thermal processing in the presence of lactate and amino acids can produce taste-active N-lactoyl amino acids. This study aimed to investigate the impact of lactate and thermal processing on the sensory characteristics of acid-hydrolyzed vegetable proteins (aHVP). The results showed that the processed aHVP exhibited enhanced kokumi, a milder umami taste, and reduced bitterness on treatment with 1% lactate at 110 °C for 3 h or 3% lactate at 120 °C for 2 h compared to the unprocessed samples. Partial or orthogonal least-squares discriminant analysis and variable importance in projection (VIP) analyses revealed the significant contributions of N-,l-Lac-l-hydrophobic AAs [-Met, -Ile, -Leu, -Val, and -Phe (VIP > 1.2)] to the observed differences between the processed and unprocessed samples. Electronic tongue analysis confirmed the sensory findings and indicated a decrease in the aftertaste of bitterness in the processed samples. Furthermore, the study identified the sensory characteristics of N-l-Lac-l-Met, -Ile, and -Leu, highlighting their potential to enhance salty, umami, and kokumi perception in simulated broth. Furthermore, the study incorporated the addition of bitter amino acids (Val, Ile, Leu, Tyr, Phe, Lys, His, and Arg) and the aforementioned N-l-Lac-l-AAs to aHVP, providing further evidence for their contributions to bitterness and aftertaste-B as well as the kokumi differences, respectively. This study provides valuable insights into the sensory effects of lactate and thermal processing on aHVP, facilitating the development of improved taste-enhancing strategies.

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.

Kokumi-Enhancing Mechanism of N-l-lactoyl-l-Met Elucidated by Sensory Experiments and Molecular Simulations.

Recent research indicates that N-lactoyl amino acid derivatives have the potential as kokumi substances, with their kokumi profile closely linked to that of amino acids. This study aimed to explore the unexplored effects resulting from the introduction of lactate groups into l-Methional (l-Met), a prevalent flavor compound found in foods, such as tomatoes, known for its ability to activate the monosodium glutamate response. N-l-Lac-l-Met was enzymatically synthesized using food grade, and its taste profile and underlying mechanisms were investigated. The structure of N-l-Lac-l-Met was determined by high-performance liquid chromatography (HPLC)-mass spectrometry (MS)/MS. Sensory evaluation revealed the presence of astringency, kokumi, and bitterness of N-l-Lac-l-Met. In a stimulated broth, N-l-Lac-l-Met exhibited enhanced umami and kokumi taste perception compared to l-Met while demonstrating good stability within pH 5 to 9. A molecular simulation and quantum mechanics analysis indicated that the formation of an amide bond played a crucial role in the kokumi-enhancing effect of N-l-Lac-l-Met, specifically by increasing its affinity with umami receptors T1R1-T1R3 and a kokumi receptor CaSR. These findings established the relationship between amide bond formation and the kokumi-enhancing effect of N-l-Lac-l-Met, presenting its potential application as the kokumi substance in the food industry.

Improved protein production in yeast using cell engineering with genes related to a key factor in the unfolded protein response.

The yeast Saccharomyces cerevisiae is a widely used cell factory for protein production. Increasing the protein production capacity of a yeast strain may be beneficial for obtaining recombinant proteins as a product or exerting its competence in consolidated bioprocessing. However, heterologous protein expression usually imposes stress on cells. Improving the cell's ability to cope with stress enhances protein yield. HAC1 is a key transcription factor in the unfolded protein response (UPR). In this study, several genes related to the UPR signal pathway, including unfolded protein sensing, HAC1 mRNA splicing, mRNA ligation, mRNA decay, translation, and Hac1p degradation, were selected as targets to engineer yeast strains. The final engineered strain produced α-amylase 3.3-fold, and human serum albumin 15.3-fold, greater than that of the control strain. Key regulation and metabolic network changes in the engineered strains were identified by transcriptome analysis and physiological characterizations. This study demonstrated that cell engineering with genes relevant to the key node HAC1 in UPR increased protein secretion substantially. The verified genetic modifications of this study provide useful targets in the construction of yeast cell factories for efficient protein production.

Six categories of amino acid derivatives with potential taste contributions: a review of studies on soy sauce.

During the fermentation of soy sauce, the metabolism of microorganisms and the Maillard reaction produce a wide variety of metabolites that contribute to the unique and rich flavor characteristics of soy sauce, such as amino acids, organic acids and peptides. Amino acid derivatives, a relatively new taste compounds, formed by the reaction of enzymes or non-enzymes from sugars, amino acids, and organic acids released through metabolism by microorganisms during soy sauce fermentation, have begun to gain more and more attention in recent years. This review focused on our existing knowledge of the sources, taste characteristics and synthesis methods of the 6 categories of amino acid derivatives, including Amadori compounds, γ-glutamyl peptides, pyroglutamyl amino acids, N-lactoyl amino acids, N-acetyl amino acids and N-succinyl amino acids. Sixty-four amino acid derivatives were detected in soy sauce, of which 47 were confirmed to have potential contribution to the taste of soy sauce, especially umami and kokumi, and some of them also have the effect of reducing bitterness. Furthermore, some amino acid derivatives, like γ-glutamyl peptides and N-lactoyl amino acids, were found to be synthesized enzymatically in vitro, which laid the foundation for further study on their formation pathways in the future.

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.

Discovery of N-l-Lactoyl-l-Trp as a Bitterness Masker via Structure-Based Virtual Screening and a Sensory Approach.

N-Lactoyl-amino acid derivatives (N-Lac-AAs) are of increasing interest as potential taste-active compounds. The complexity and diversity of N-Lac-AAs pose a significant challenge to the effective discovery of taste-active N-Lac-AAs. Therefore, a structure-based virtual screening was used to identify taste-active N-Lac-AAs. Virtual screening results showed that N-lactoyl-hydrophobic amino acids had a higher affinity for taste receptors, specifically N-l-Lac-l-Trp. And then, N-l-Lac-l-Trp was synthesized in yields of 22.3% by enzymatic synthesis in the presence of l-lactate and l-Trp, and its chemical structure was confirmed by MS/MS and one-dimensional (1D) and two-dimensional (2D) NMR. Sensory evaluation revealed that N-l-Lac-l-Trp had a significant taste-masking effect on quinine, d-salicin, caffeine, and l-Trp, particularly l-Trp and caffeine. N-l-Lac-l-Trp had a better masking effect on the higher concentration of bitter compounds. It reduced the bitterness of caffeine (500 mg/L) and l-Trp (1000 mg/L) by approximately 20 and 26%, respectively. The result of the ligand-receptor interaction and a quantum mechanical analysis showed that N-l-Lac-l-Trp increased the binding affinity to the bitter receptor mainly through hydrogen bonding and lowering the electrostatic potential.

Quantitative studies, taste recombination, and omission experiments on the key taste compounds in Chinese and Japanese soy sauce.

The non-volatile compounds in Chinese soy sauce (CSS) and Japanese soy sauce (JSS) were qualitatively and quantitatively identified by amino acid analyser and gas-chromatography mass-spectrometry combined with derivatization method (DER-GC-MS). Forty-nine compounds were identified, including 19 amino acids, 8 sugars, 6 sugar alcohols, 8 organic acids and 8 others. Six groups of taste compounds were added to the water at natural concentration in soy sauce were able to reconstitute taste models of CSS and JSS, with no remarkable differences from the soy sauce taste profiles. Through three different rounds of omission tests, it was clear that the recombination models of 10 and 11 key compounds could simulate the typical taste characteristics of CSS and JSS, respectively, with the shared compounds NaCl, glutamate, lysine, isoleucine, leucine, lactic acid, citric acid, and > 500 Da components. This study provided an important theoretical basis for the in-depth research on the soy sauce flavor.

Comprehensive Analysis of Signal Peptides in Saccharomyces cerevisiae Reveals Features for Efficient Secretion.

Signal peptides (SPs) are N-terminus sequences on the nascent polypeptide for protein export or localization delivery, which are essential for maintaining cell function. SPs are also employed as a key element for industrial production of secreted recombinant proteins. Yet, detailed information and rules about SPs and their cellular interactions are still not well understood. Here, systematic bioinformatics analysis and secretion capacity measurement of genome-wide SPs from the model organism Saccharomyces cerevisiae is performed. Several key features of SPs, including region properties, consensus motifs, evolutionary relationships, codon bias, e.g., are successfully revealed. Diverse cell metabolism can be trigged by using different SPs for heterologous protein secretion. Influences on SPs with different properties by chaperones can cause different secretory efficiencies. Protein secretion by the SP NCW2 in SEC72 deletion strain is 10 times than the control. These findings provide insights into the properties and functions of SPs and contribute to both fundamental research and industrial application.

Flavor differences of soybean and defatted soybean fermented soy sauce and its correlation with the enzyme profiles of the kojis.

BACKGROUND: Soybeans and defatted soybeans, commonly used as protein ingredients, have different flavors of their fermented soy sauce. Clarifying the differences between the two soy sauces, as well as the formation mechanism, is an important prerequisite for improving the flavor of defatted soybean soy sauce. To this goal, the aroma characteristics of two soy sauces and their volatile profiles were compared by sensory evaluation and gas chromatography-mass spectrometry, and eight enzyme activities and volatile profiles of matured koji were determined. RESULTS: Sensory results showed that the acids, fruity and cooked potato-like attributes were higher in whole soybean fermented soy sauce, whereas defatted soybean soy sauce exhibited higher smoky and malty attributes, closely related to the contents of aroma-active compounds in soy sauce, such as isobutyl acetate, 2/3-methylbutanal, acetic acid and 2/3-methylbutanoic acid. The content of most volatiles in the matured kojis showed a consistent trend with that of soy sauce: alcohols, acids, furan(one)s and ketones. Interestingly, acid protease and cellulase activities were 3.3 and 1.6 times higher in the whole soybean koji than in defatted soybean koji, respectively, whereas neutral protease, aminopeptidase, glucoamylase and ß-glucosidase were approximately 2.0 times higher in defatted soybean koji. CONCLUSION: In summary, the flavor differences between soybean and defatted soybean fermented soy sauce were not only caused by the differences in the content of flavor precursors in the materials, but also closely related to the differences in the enzymatic profiles accumulated during the koji-making process. © 2022 Society of Chemical Industry.

Secretion of collagenases by Saccharomyces cerevisiae for collagen degradation.

BACKGROUND: The production and processing of animal-based products generates many collagen-rich by-products, which have received attention both for exploitation to increase their added value and to reduce their negative environmental impact. The collagen-rich by-products can be hydrolyzed by collagenases for further utilization. Therefore, collagenases are of benefit for efficient collagen materials processing. An alternative and safe way to produce secreted collagenases is needed. RESULTS: Two collagenases from Hathewaya histolytica, ColG and ColH, were successfully secreted by the yeast Saccharomyces cerevisiae. Compared with the native signal peptide of collagenase, the α-factor leader is more efficient in guiding collagenase secretion. Collagenase secretion was significantly increased in YPD medium by supplementing with calcium and zinc ions. Recombinant collagenase titers reached 68 U/mL and 55 U/mL for ColG and ColH, respectively. Collagenase expression imposed metabolic perturbations on yeast cells; substrate consumption, metabolites production and intracellular cofactor levels changed in engineered strains. Both recombinant collagenases from yeast could hydrolyze soluble and insoluble collagen materials. Recombinant ColG and ColH showed a synergistic effect on efficient collagen digestion. CONCLUSIONS: Sufficient calcium and zinc ions are essential for active collagenase production by yeast. Collagenase secretion was increased by optimization of expression cassettes. Collagenase expression imposed metabolic burden and cofactor perturbations on yeast cells, which could be improved through metabolic engineering. Our work provides a useful way to produce collagenases for collagen resource utilization.

Community structure of yeast in fermented soy sauce and screening of functional yeast with potential to enhance the soy sauce flavor.

Yeast plays an important role in the formation of desirable aroma during soy sauce fermentation. In this study, the structure and diversity of yeast communities in seven different soy sauce residues were investigated by ITS sequencing analysis, and then the aroma characteristics of selected yeast species were examined by a combination of gas chromatography-mass spectrometry (GC-MS), headspace solid-phase microextraction (SPME) and liquid-liquid extraction (LLE). A total of 18 yeast genera were identified in seven soy sauce residues. Among them, Candid and Zygosaccharomyces were detected in all samples, followed by Millerozyma, Wickerhamiella, Meyerozyma, Trichosporon and Wickerhamomyces, which were found in more than two-thirds of the samples. Subsequently, eight representative species, isolated from soy sauce residues, were subjected to environmental stress tolerance tests and aroma production tests. Among them, three isolated species were regarded as potential aroma-enhancing microbes in soy sauce. Wickerhamiella versatilis could increase the contents of ethyl ester compounds and alcohols, thereby improving the fruity and alcoholic aroma of soy sauce. Candida sorbosivorans enhanced sweet and caramel-like aroma of soy sauce by producing 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 3-hydroxy-2-methyl-4h-pyran-4-one (maltol). Starmerella etchellsii could enhance the contents of 2,6-dimethylpyrazine, methyl pyrazine and benzeneacetaldehyde. This study is of great significance for the development and application of flavor functional yeasts in soy sauce fermentation.