Molecular Microbiology Research 2025, Vol.15 http://microbescipublisher.com/index.php/mmr © 2025 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher.
Molecular Microbiology Research 2025, Vol.15 http://microbescipublisher.com/index.php/mmr © 2025 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Publisher MicroSci Publisher Editedby Editorial Team of Molecular Microbiology Research Email: edit@mmr.microbescipublisher.com Website: http://microbescipublisher.com/index.php/mmr Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Molecular Microbiology Research (ISSN 1927-5595) is an open access, peer reviewed journal published online by MicroSci Publisher. The journal publishes all the latest and outstanding research articles, letters and reviews in all areas of molecular microbiology, including original articles, reviews and brief reports in microbiology, bacteriology, mycology, molecular and cellular biology and virology at the level of gene expression and regulation, genetic transfer, cell biology and subcellular organization, pathogenicity and virulence, physiology and metabolism, cell-cell communication and signalling pathways as well as the interactions between the various cell systems of microorganisms including the interrelationship of DNA, RNA and protein biosynthesis. All the articles published in Molecular Pathogens are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MicroSci Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights. MicroSci Publisher is an international Open Access publisher specializing in microbiology, bacteriology, mycology, molecular and cellular biology and virology registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada.
Molecular Microbiology Research (online), 2025, Vol. 15, No. 2 ISSN 1927-5595 http://microbescipublisher.com/index.php/mmr © 2025 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Latest Content From Northern Song Heritage to Modern Innovation: A Study on the Technological Evolution and Quality Control of Yuyue's Traditional Sauce-Making Craft Xudong Chen, Zeqin Chen, Yelin Huang, Jinghong Wang, Lei Yong Molecular Microbiology Research, 2025, Vol. 15, No. 2, 45-58 Research Progress and Trends in Early-Maturing, High-Quality, and Stress-Resistant Breeding Techniques for Potato Tianxia Guo, Xingde Wang Molecular Microbiology Research, 2025, Vol. 15, No. 2, 59-68 A Comprehensive Review of Genetic Basis and Breeding Strategies for Enhancing Cotton Resistance to Fungal Diseases Jin Zhou, Jiong Fu Molecular Microbiology Research, 2025, Vol. 15, No. 2, 69-81 Genetic Management of Major Rice Diseases Haiying Huang, Danyan Ding Molecular Microbiology Research, 2025, Vol. 15, No. 2, 82-92 From Microbes to Medicines: Strategy, Carrier Design, and the Hard Problem of Targeted Delivery Congbiao You , Xing Zhao Molecular Microbiology Research, 2025, Vol. 15, No. 2, 93-99
Molecular Microbiology Research, 2025, Vol.15, No.2, 45-58 http://microbescipublisher.com/index.php/mmr 45 Research Report Open Access From Northern Song Heritage to Modern Innovation: A Study on the Technological Evolution and Quality Control of Yuyue's Traditional Sauce-Making Craft Xudong Chen, Zeqin Chen, Yelin Huang, Jinghong Wang, Lei Yong Lishui Yuyue Brewing Food Co., Ltd, Lishui, 323006, Zhejiang, China Corresponding author: 465362494@qq.com Molecular Microbiology Research, 2025, Vol.15, No.2 doi: 10.5376/mmr.2025.15.0006 Received: 10 Jan., 2025 Accepted: 22 Feb., 2025 Published: 10 Mar., 2025 Copyright © 2025 Chen et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Chen X.D., Chen Z.Q., Huang Y.L., Wang J.H., and Yong L., 2025, From Northern Song heritage to modern innovation: a study on the technological evolution and quality control of Yuyue's traditional sauce-making craft, Molecular Microbiology Research, 15(2): 45-58 (doi: 10.5376/mmr.2025.15.0006). Abstract The soy sauce brewing technique passed down from the Northern Song Dynasty was reborn in the context of modern food industry. This study takes the traditional soy sauce making technology of the century-old brand "Yuyue" in Lishui, Zhejiang, as the object, and studies its process evolution and quality control methods. Tracing the origin of Yuyue soy sauce technology, it explains the process from the inheritance of natural brewing methods in the Northern Song Dynasty and Qinguan period to the establishment of soy sauce gardens in the Republic of China and the reform of modern enterprises. Through the analysis of the microbial fermentation system, the trend of soy sauce fermentation gradually transitioning from natural inoculation to breeding bacterial species is revealed, and the mechanism of action of major functional microorganisms (Aspergillus, lactic acid bacteria, yeast, etc.) in the formation of flavor substances is clarified. This study will further explore the effect of process optimization based on microbial regulation, such as bacterial strain improvement, mixed fermentation and intelligent control on improving the flavor of soy sauce and shortening the fermentation cycle, and then build a quality evaluation system, including chemical index determination, sensory assessment and standardization processes, and analyze food safety control strategies to prevent and control risks such as bioamines. Research shows that the integration of traditional brewing and modern technology can improve the consistency of production efficiency and quality while ensuring the unique flavor and safety of soy sauce. In order to ensure the experience of the fish-leap sauce making process in inheritance and innovation, it can provide a reference for the modernization of traditional fermented foods. Keywords Soy sauce fermentation; Microorganisms; Flavor formation; Quality control; Food safety; Intelligent brewing 1 Introduction As a fermented condiment with a long history, soy sauce has a production tradition in China for more than two thousand years. The "fish leap" soy sauce brewing technique in Lishui, Zhejiang is said to be derived from the legacy of the Northern Song Dynasty, reflecting the continuation and development of ancient sauce making. The Yuyue brand was founded in 1919. Its predecessor was Desheng Sauce Garden, which is a famous century-old brand in the local area. After several generations of inheritance, the Yuyue soy sauce brewing technique was included in the Lishui City Intangible Cultural Heritage List in the 2000s, becoming a representative of traditional brewing with regional characteristics. Yuyue Soy Sauce insists on natural fermentation of soybeans, wheat, salt and other raw materials, and follows traditional methods such as steaming beans in wooden barrels and fermenting in open air for more than 180 days. The products are known for their rich sauce flavor and mellow aftertaste. Traditional small workshop brewing also faces challenges such as long fermentation cycle, unstable quality and difficulty in sanitation control. Under the conditions of the contemporary food industry, how to combine ancient sauce-making experience with modern technology to achieve process optimization and quality standardization has become a research hotspot. In recent years, advances in microbiology and fermentation engineering have allowed us to deeply analyze the mechanism of action of microbial communities during soy sauce fermentation (Yang et al., 2017). By selecting and breeding high-performance bacterial strains, applying intelligent control of
Molecular Microbiology Research, 2025, Vol.15, No.2, 45-58 http://microbescipublisher.com/index.php/mmr 46 fermentation conditions, and establishing objective flavor and quality evaluation indicators, traditional soy sauce brewing is bursting with new vitality (An et al., 2023). In this context, this study takes the traditional sauce making technique of Yuyue as an example to systematically explore its process evolution, microbial fermentation system, flavor formation mechanism, process optimization and intelligent path, quality evaluation standards and food safety control, analyze the practical effects of the integration of tradition and innovation, and look forward to the future development direction. The research aims to provide scientific basis and typical demonstration for the modernization and upgrading of the traditional fermented food industry. 2 The Origin and Evolution of Traditional Sauce Making Techniques of Fish Leap 2.1 Historical origin: inheritance of the legacy of Northern Song Dynasty The brewing technique of Yuyue soy sauce is said to originate in the Northern Song Dynasty, and corresponds to the method of making sauces promoted by historical figures such as Qin Guan in Lishui. This ancient method takes natural fermentation as the core, and relies on microorganisms that naturally exist in the environment to make koji and ferment, without using artificial inoculated purebred bacteria. During the Yuan, Ming and Qing dynasties, folk experience in hand-made soy sauce was accumulated continuously and passed down from region to region. The ancient recipes and processes of Yuyue Soy Sauce are the crystallization of this historical heritage. In 1919, Ying Desheng founded the "Desheng Sauce Garden" in Lishui, marking the beginning of the Yuyue brand. The establishment of Desheng Sauce Garden not only inherited the traditional soybean skills, but also introduced the advanced management concepts at that time, realizing the transition from manual skills to commercial production. Since then, several generations of descendants have abide by the ancient methods and strive for excellence, making Yuyue Soy Sauce famous in the local area. The origin of the fish leap technique reflects the context of Chinese soy sauce brewing from the interests of court literati to the development of folk handicrafts, and also demonstrates the tenacious vitality of intangible cultural heritage techniques after dynasties. 2.2 Craft features: traditional brewing process Yuyue’s traditional sauce making technique has strict and sophisticated process, representing the essence of the ancient methods of Jiangnan sauce garden. Its main raw materials are soybeans and flour. The brewing process includes soaking beans, steaming beans in wooden barrels, cooling, natural inoculation of mold, hand-made koji, pressing koji mash (that is, mixing the cultivated koji with cooked salt water to form sauce mash), long-term fermentation of open-air sun-drying, pressing sauce, torture and concentration, and sealing and aging of the jar. Among them, "three fermentation, second immersion, and one pressing" is the unique process key of fish leaping soy sauce, which means supplementing nutrients and prolonging fermentation in stages during the long fermentation process to give full play to the role of microbials (Duan et al., 2010). It is particularly worth mentioning that the open-air fermentation process: the fish leaps in large outdoor tanks for sun exposure for at least 180 days. This open long-term fermentation allows Aspergillus, yeast, bacteria, etc. to reproduce and metabolize in different seasons, giving the soy sauce a rich and mellow flavor (Figure 1) (Yang et al., 2017). After pressing the raw butter, Yuyue Soy Sauce insists on "frying soy sauce" on low heat, that is, heat and concentrate the soy sauce to a certain baume, which not only kills the remaining bacteria to ensure safety, but also adds the sauce color and aroma through the Melade reaction. The ancient fish-leap craftsmanship has strict requirements for every detail, and the brewing master needs to judge the temperature and timing based on experience. Its products have traditional characteristics of rich sauce flavor, salty, fresh and palatable, and reddish brown and transparent color, which are very popular among consumers. 2.3 Modern inheritance and innovation Entering the 21st century, the old Yuyue Soy Sauce factory once faced the challenge of market competition and decline in efficiency. In 2006, with the support of local governments, Yuyue carried out enterprise restructuring, and the tenth-generation successor Chen Xudong took over the sauce garden. He adheres to the concept of "not seeking the top 100, but seeking the top 100 years", insists that the core of ancient brewing remains unchanged,
Molecular Microbiology Research, 2025, Vol.15, No.2, 45-58 http://microbescipublisher.com/index.php/mmr 47 and at the same time injects modern innovation elements (Liu et al., 2024). Yuyue established a provincial brewing technology research and development center and began to scientifically record and improve traditional processes. In recent years, Yuyue has launched a series of new products that retain both traditional flavors and meet modern needs, such as strengthened soy sauce with iron added, healthy soy sauce with reduced salinity, and orange vinegar and lotus seed sauce wine developed in combination with local specialties. In terms of process equipment, Yuyue invested in the transformation of sauce drying yards and fermentation containers, introduced facilities such as stainless steel pipe temperature control drying yards to partially replace manual labor, and at the same time, it combined traditional skills with industrial tourism through glass observation windows and model displays. Figure 1 Field view of traditional soy sauce fermentation using earthenware vats in natural environments 3 Microbial Fermentation System: from Natural Inoculation to Bacterial Breeding 3.1 Traditional sauce-based microbial community Traditional soy sauce fermentation is a complex microbial synergistic process carried out in an open natural environment. In the ancient method of fish jumping, artificial bacteria are not actively added during the rumbling process, but instead use mold spores that naturally exist in the environment to inoculate them by themselves. After several days of indoor incubation under warm conditions, the soybean-bran mixture becomes covered with fluffy Aspergillus mycelia, forming what is known as “jiangqu” (fermentation starter). Research has shown that under traditional conditions, fungi of the genus Aspergillus, such as Aspergillus oryzae, often dominate during the koji-making stage. These fungi produce amylases and proteases, which break down macromolecules in soybeans and wheat flour into fermentable substrates (Wang et al., 2023). When the koji is mixed with brine to initiate high-salt moromi fermentation, the environment—characterized by approximately 20% salt concentration, high humidity, and low oxygen—favors the enrichment of halotolerant microbial communities. Lactic acid bacteria (e.g., Tetragenococcus halophilus) and yeasts (e.g., Zygosaccharomyces rouxii) are the major functional microbial groups in traditional moromi fermentation, each becoming dominant at different fermentation stages (Chin et al., 2024). In the natural fermentation system since the Northern Song Dynasty, the source of microorganisms completely relies on the environment and raw materials, and naturally select bacterial species that are suitable for the conditions of the soybean mash. Research on traditional Korean soy sauce also found that under the conditions of adding sterile species, communities composed of various bacteria and fungi will be spontaneously formed, including salt-resistant lactic acid bacteria, Pseudomonasaceae and a small amount of yeast, etc., which work together to form the flavor and quality of soy sauce. 3.2 Pure breeding fermentation and breeding of bacteria With the development of modern microbiology, the soy sauce industry has begun to introduce the concept of purebred fermentation, that is, artificial inoculation of specific excellent bacterial strains to replace natural fermentation that is completely dependent on the environment (Liu et al., 2025). As early as the late 19th century, Japan isolated and purified excellent Aspergillus and used it for soy sauce koji, achieving the stability of purebred
Molecular Microbiology Research, 2025, Vol.15, No.2, 45-58 http://microbescipublisher.com/index.php/mmr 48 koji. Modern large soy sauce factories generally use selected Aspergillus oryzae strains to make koji to ensure consistent enzyme activity and fermentation performance of the koji. At the same time, during the high-salt fermentation stage, some companies also add purely cultured functional bacteria, such as inoculating mixed bacterial agents with salt-resistant lactic acid bacteria and yeast in the sauce mash to speed up fermentation and inhibit miscellaneous bacteria. Compared with natural inoculation, artificially domesticated bacteria have the advantages of strong fermentation, high enzyme production and safe and controllable. In recent years, scientists have further carried out bacterial breeding work and cultivated soy sauce fermented strains with optimized performance. For example, studies have successfully screened out salt-resistant lactic acid strains with low-yield acetic acid and used for soy sauce fermentation to reduce the inhibition of acetic acid on yeast and improve the fermentation alcohol production and flavor quality. Qi et al. (2021) reported a T. halophilus mutant strain that lacks the key enzyme of the citric acid metabolism pathway, which does not produce excessive acetic acid. The use of this bacterial fermentation makes the alcohol fermentation of Z. rouxii yeast more vigorously, and the soy sauce produced has a more fragrant and lower sour taste (Qi et al., 2021). This shows that through strain breeding and improvement, certain fermentation characteristics can be improved in a directional manner, thereby optimizing the flavor and quality of soy sauce. 3.3 Microbial diversity and synergistic interactions Whether traditional natural fermentation or modern inoculation fermentation, soy sauce brewing is the result of the joint action of multiple microorganisms. In the early stage of fermentation, aerobic fungi such as Aspergillus multiply in large quantities, providing saccharification and protein decomposition products; then, under high-salt anaerobic conditions, halophilic lactic acid bacteria reproduce in large quantities, fermenting some sugars to produce acids (such as lactic acid), reducing the pH of the mash, creating conditions for subsequent yeast activities.Studies have shown that in naturally fermented moromi at room temperature, lactic acid bacteria and Bacillus species are actively involved in acid production during the initial phase. In contrast, temperature-controlled fermentation tends to enrich more lactic acid bacteria, leading to a more stable acidification process. In the mid to late stages, salt-tolerant yeasts such as Zygosaccharomyces rouxii utilize the sugars and amino acids generated to carry out alcoholic and ester fermentations, producing alcohols and esters that contribute to the aromatic profile of the soy sauce (Devanthi et al., 2018). At the same time, facultative anaerobic bacteria (e.g., Staphylococcus spp.) continue to degrade proteins, releasing amino acids and small peptides that enhance the umami taste. During open-air fermentation processes, diurnal temperature variations and seasonal changes also influence the succession of dominant microbial communities. High summer temperatures favor aroma-producing yeasts, while colder winter conditions help preserve moromi by supporting psychrotolerant bacteria. Through genomic and transcriptomic analyses, Chun et al. (2021) further revealed that in the natural fermentation of traditional Korean soy sauce, anaerobic glycolysis by lactic acid bacteria is the dominant metabolic pathway. Meanwhile, some aerobic halophilic bacteria (such as Chromohalobacter and Halomonas) participate in the oxidative metabolism of amino acids and other compounds. Yeasts (e.g., Debaryomyces, Wickerhamomyces) are closely associated with the production of biogenic amines and specific flavor compounds. 4 Formation Mechanism and Regulation Strategy of Flavor Substances 4.1 Analysis of main flavor ingredients The reason why soy sauce has a unique taste is due to the combined action of a large number of flavored substances and aromatic compounds produced during the fermentation process. Its basic salty and fresh flavor comes from the superposition of high-salt environments and amino acids. Among them, umami flavor (quality) is mainly contributed by amino acids and small peptides, especially the high content of free amino acids such as glutamic acid and aspartic acid, which will bring umami flavor and thick flavor. When soy sauce fermentes, the protein deteriorates in the synergistic action of Aspergillus protease and bacterial enzymes, releasing more than 20 free amino acids and short peptides, forming a nitrogen source substance rich in soy sauce. In addition,
Molecular Microbiology Research, 2025, Vol.15, No.2, 45-58 http://microbescipublisher.com/index.php/mmr 49 nucleotides (such as inosine and guanylic acid) also come from microbial cell lysis and show "umami-taste synergy" with amino acids (Huang et al., 2023). The salty taste is mainly provided by table salt, and high salinity not only gives salty taste but also a necessary condition for antibacterial. Sweetness and bitterness are weaker, and are contributed by some amino acids (sweet glycine, bitter leucine), sugars, bitter peptides, etc., but they are not prominent in the complex flavor of soy sauce. The aroma components of soy sauce are very complex, with hundreds of volatile organic compounds, but the overall aroma can be described as a coordinated combination of soy sauce (fermented black beans), burnt aroma and fruit vinegar aroma. The main aroma substances include: alcohols, organic acids, esters, phenols, furones, pyrazines and sulfides. The sources of these volatiles vary: alcohols, acids, esters, etc. are mainly produced by fermentation and metabolism of yeast and bacteria; most of them are the products of Maillard reaction and caramelization reaction, and are produced by reaction of amino acids and sugars during the long-term fermentation and later heating and concentration of the sauce mash. Especially the "frying soy sauce" step in the fish jumping process, gentle heating causes the residual sugar to react with the amino acids to Maillard, which significantly enhances the rich soy sauce aroma and attractive brown-red luster of the soy sauce. 4.2 The role of microorganisms in flavor formation The metabolic activities of microorganisms determine the types and quantities of flavor compounds produced during soy sauce fermentation. Molds (Aspergillus spp.) secrete hydrolytic enzymes that break down non-flavor macromolecular substrates (e.g., starch and proteins) into flavor precursors. These precursors not only contribute directly to the basic sweet and umami taste of soy sauce but also serve as substrates for subsequent microbial fermentation. In the early fermentation stage, lactic acid bacteria ferment part of the sugars into lactic acid and trace amounts of acetic acid. The mild acidity of lactic acid enhances flavor complexity and lowers the pH, thereby inhibiting undesirable microorganisms. Additionally, lactic acid bacteria produce certain volatile short-chain fatty acids, contributing to the fatty and mellow aroma of soy sauce. Furthermore, yeasts—particularly salt-tolerant species such as Zygosaccharomyces rouxii—are considered major contributors to soy sauce aroma. Yeasts ferment sugars into ethanol, which can react with organic acids to form various esters (e.g., ethyl acetate and ethyl lactate), imparting fruity and alcoholic aromas (Wu et al., 2022). Yeast also produces ester and phenolic aromatic substances through higher alcohol metabolism, such as 4-ethylguaia lignol, and so on in soy sauce smoked incense. However, different microorganisms have a synergistic effect on flavor: the ethanol produced by yeast can further react with the organic acid produced by lactic acid bacteria to form esters and enrich aroma; the large amount of amino acids provided by Aspergillus is more likely to undergo Maillard browning reaction to form calcified aroma substances under the conditions of lowering the pH of lactic acid bacteria (Wu and Yan, 2024). Therefore, a variety of fermented microorganisms have created a complete flavor spectrum of soy sauce. Studies have also found that certain minor microbial contaminants in traditional processes, such as species of Rhizopus and Penicillium, may positively influence flavor when present in small amounts. These microbes can produce unique enzyme systems and metabolites that enhance the aromatic complexity of soy sauce (Zhang et al., 2023). This may help explain why traditionally open-air fermented soy sauce often exhibits richer flavor profiles than those fermented in fully enclosed pure-culture systems. 4.3 Flavor control and process parameter optimization In modern soy sauce brewing, in order to obtain stable and excellent flavor, the key influencing factors in the fermentation process need to be regulated. The first is the fermentation temperature. Temperature not only affects the reaction rate, but also directly changes the composition of metabolites. A study of Bacillus subtilis related to soy sauce was cultivated at different temperatures. The results showed that at 37 ℃, the bacteria metabolize more ammonia odor substances, while above 45 ℃, more soy sauce aroma substances were generated. In actual production, appropriate high temperatures of soybean paste (such as 35 °C~40 °C) are beneficial to inhibit odor production and promote yeast fragrance production, while ripening at low temperatures in the later stage can reduce the accumulation of pungent odor (Gao et al., 2011).
Molecular Microbiology Research, 2025, Vol.15, No.2, 45-58 http://microbescipublisher.com/index.php/mmr 50 Second, the effect of salt concentration on flavor is significant: high salt can inhibit the growth of mixed bacteria to ensure clean soy sauce, but at the same time, excessive salinity slows down the fermentation process and the formation of fragrance substances. To balance safety with flavor, traditional high-salt fermentation (about 18%~20% salt) has a long cycle but a strong flavor; in modern times, low-salt soy sauce processes (such as 12%~16% salt) have emerged, and the fermentation speed is accelerated but if no measures are taken, the flavor may become lighter (Kim et al., 2020). In this regard, industrial exploration has been conducted to add salt substitutes (such as KCl partially replace NaCl) or extend brewing time to compensate for the adverse effects of reducing salt on flavor. Third, the fermentation time directly determines the accumulation of flavor substances. Generally speaking, longer fermentation and maturation times contribute to the formation of complex flavors. NMR metabolomics monitoring found that as fermentation extends from 0 to 8 months, key flavor precursors such as ethanol and amino acids in soy sauce continue to rise, and aroma substances form inflection points after fermentation for about 90 days (Figure 2). Therefore, traditionally, soy sauce is best fermented for at least six months. For industrial applications that want to shorten the cycle, it is possible to consider accelerating the reaction by increasing the temperature, adding enzyme preparations or inoculating highly active bacterial strains, but a certain maturity time is still required to generate the later fragrance. Fourth, strain matching and inoculation strategies are also flavor regulation methods. If segmented inoculation is used: first inoculate lactic acid bacteria and ferment for a certain period of time, then inoculate yeast (sequential inoculation), it can avoid competition between the two and improve the yeast fragrance production efficiency. Figure 2 Impact of temperature, humidity, light exposure, and lid sealing on flavor compound accumulation during open fermentation 5 Optimization and Intelligent Path of Sauce Making Process Based on Microbial Regulation 5.1 Space-time dynamic monitoring and regulation of dominant bacterial species activity The core idea of process optimization on a traditional basis is to give full play to the greatest role of beneficial microorganisms and inhibit adverse factors. Breeding of excellent bacterial species is the prerequisite for process optimization. For example, obtaining high-yield enzyme-producing Aspergillus oryzae strains through mutagenesis breeding or genetic engineering can significantly improve the efficiency of koji production, shorten the time of koji production and increase the saccharification force, and thus accelerate the subsequent fermentation speed. Secondly, use the fermentation strategy of compound bacterial strains: traditional fermentation relies on the natural formation of mixed communities, while modern fermentation can actively construct combinations as needed. By inoculating Aspergillus, lactic acid bacteria, and yeasts in optimal proportions, multiple metabolic pathways can be activated simultaneously, thereby accelerating fermentation while preventing the overgrowth of any single strain that could lead to off-flavors. In addition, some continuous or intermittent stirring and fermentation processes have also been introduced into soy sauce production. For example, Japan once developed stir tanks to continuously ferment, reducing the fermentation cycle from half a year to several weeks. However, the continuous fermentation flavor is not as mellow as the stand-alone fermentation, and is currently mostly used as a partial replacement process. Combined
Molecular Microbiology Research, 2025, Vol.15, No.2, 45-58 http://microbescipublisher.com/index.php/mmr 51 with modern biotechnology, factories can choose fermentation mode according to product needs: for quick brewing soy sauce, inoculation + temperature control + stirring can be used; for high-end soy sauce, long-term static fermentation is adhered to, supplemented by excellent bacterial strains, the products of the two will differ in indicators such as amino acid nitrogen and aroma substances. 5.2 Artificial intervention strategies for microecosystems in fermentation environments In the era of Industry 4.0, intelligent control technology has gradually been applied to the traditional fermentation industry, and soy sauce brewing has also begun to explore digital and automated production paths. Traditional soy sauce gardens rely on the experience of brewing masters to judge the fermentation status, such as distinguishing the aroma of the mash through smell, visual observation of bacterial film and liquid color, and judging the heating nodes by hand touching the temperature. Although these experiences are valuable, they are subjective and difficult to standardize. To this end, modern factories have introduced various sensors and online analytical instruments to monitor fermentation parameters in real time to judge microbial fermentation activity. When an abnormality is detected, such as excessive temperature, Aspergillus may autolysis or lactic acid bacteria may die from overheating, the system can automatically alarm and start the corresponding adjustment device (such as starting the cooling water cycle or stopping heating). Through the Internet of Things (IoT), traditional sauce jars can also be connected to digital systems to realize remote data collection and control (Chen, 2013). Another intelligent direction is applied machine learning and big data analysis. Enterprises can collect multi-dimensional data (temperature, bacterial phase, chemical composition, etc.) of different batches of soy sauce during the fermentation process, and combine the final quality evaluation results to train the prediction model. Some large soy sauce factories in Japan have partially realized automation: from raw material cooking, koji stirring, fermentation and stirring to pressing, sterilization and packaging, all links are performed by programmable controllers (PLCs) and robots, achieving stable mass production. However, for brands such as Yuyue, which adhere to the ancient method, dual monitoring of artificial sensors and instruments will still be adopted during the key flavor formation stage, and adjustments will be made with caution. In intelligent transformation, how to avoid destroying traditional flavors is an important consideration. For example, the introduction of automatic opening and closing sheds in the open-air sunlight process can control sunshine and rainfall, which can ensure the flavor while reducing the pollution of mixed bacteria and fermentation stagnation caused by the weather. For example, if the use of electronic nose and electronic tongue devices to evaluate the aroma and taste of the fermentation broth in real time, it can be used to automatically determine the fermentation progress and coordinate the temperature stirring and other parameter adjustments. The application of these intelligent equipment makes traditional experience digital, which is conducive to the inheritance of skills and training of newcomers. 5.3 Integration of microbial process modeling and brewing intelligent control system In addition to microbial and control technologies, some emerging processing technologies are also trying to integrate into the soy sauce process in order to improve quality or develop new varieties. Physical technologies such as ultrasonic processing and high-pressure processing have been studied to accelerate certain enzyme reactions or sterilization (Higuchi et al., 2024), but the impact on flavor needs to be carefully evaluated. The development of enzyme engineering has enabled industrial enzyme preparations to supplement Aspergillus enzymes and added to fermentation mash to improve protein decomposition rate, thereby obtaining soy sauce with high amino acid nitrogen content in a shorter time. However, excessive reliance on exogenous enzymes may lead to monotonous flavor and must be used reasonably. On the other hand, biotechnical means such as metabolic engineering are being used to customize fermentation strains. With advances in synthetic biology, it may even be possible in the future to "synthese" key flavor complexes of soy sauces by engineering microorganisms, thereby significantly shortening fermentation times. However, pure chemical or biological rapid production is currently difficult to reproduce the round and comprehensive flavor given by traditional fermentation. Therefore, the more realistic path at present is the combination of innovation in
Molecular Microbiology Research, 2025, Vol.15, No.2, 45-58 http://microbescipublisher.com/index.php/mmr 52 some links and overall process inheritance. For example, the iron-enhancing soy sauce developed by Yuyue is to add an appropriate amount of lactoferrin and soluble iron salt before packaging based on traditional brewing to enhance nutritional function while ensuring that the original soy aroma is not destroyed. This product innovation expands the functional attributes of traditional soy sauce. Industrial upgrading is also reflected in model innovation, such as Yuyue building the soy sauce brewing industrial park into a platform integrating production, popular science and tourism. Through modern display technology, consumers can intuitively understand the mysteries of soy sauce microbial fermentation and the value of traditional craftsmanship. This experience economy and cultural communication have enhanced the brand influence and brought new benefits to the company. 6 Construction and Standardization of Quality Evaluation System 6.1 Traditional evaluation and modern analysis The quality of soy sauce is traditionally mainly based on experienced brewing masters to identify sensory features from the aspects of color, fragrance, taste, etc. Wine reviewers usually judge the quality of soy sauce by observing its translucent color, smelling its rich and pure aroma, and savoring its freshness and mellowness. However, this sensory evaluation is subjective and is not easy to quantify and standardize. To this end, modern quality evaluation has introduced a method that combines instrument analysis with sensory science. On the one hand, the objective physical and chemical indicators of soy sauce are determined through chemical analysis: such as amino acid nitrogen, total nitrogen, salt, total acid, color, sugar, etc. High-quality soy sauce usually has an amino acid nitrogen content of more than 1.0 g/100mL and a balanced proportion, with moderate total acid and reddish-brown color. The analysis of the aroma and taste of soy sauce is increasingly relying on instruments and means. For example, gas chromatography-mass spectrometry (GC-MS) combined with odor olfactory resolution (GC-O) was used to analyze volatile aroma components; free amino acids and nucleotide composition were determined by high performance liquid chromatography (HPLC); and the umami, salty and bitterness intensity of the sample were objectively evaluated using an electronic tongue sensor (Feng et al., 2013). Through these analyses, a "chemical fingerprint" of a soy sauce sample can be obtained. Texture analysis, viscosity measurement, etc. are also used to evaluate the taste characteristics of soy sauce (such as consistency). However, the chemical analysis data is not a simple linear relationship with human sensory feelings. To this end, it is necessary to introduce a sensory evaluation team to conduct systematic evaluation and analyze the sensory results with physical and chemical indicators. Liu et al. (2023) conducted a sensory and physical and chemical analysis of aged soy sauce in different years, and found that as the maturity year increases, the total solids, amino acid nitrogen, and color of the soy sauce all increase (Liu et al., 2023), showing higher viscosity and darker color, and obtain higher thick, fresh sweet and mellow scores on the sensory. They also pointed out that consumers prefer soy sauces with dark colors, high viscosity and outstanding sweet and umami flavor. 6.2 Quality standardization and grade classification In order to unify the quality measurement standards in the industry, various countries have formulated quality standards for soy sauce. China's current soy sauce standard (GB/T 18186) divides soy sauce into special grade, first grade, second grade, etc. according to indicators such as amino acid nitrogen content. Extra-grade soy sauce requires amino acid nitrogen ≥0.8 g/100mL, and sensoryly requires coordinated color, aroma and odor without odor (Wang et al., 2018). This standard prompts brewing companies to strictly control processes in production to ensure that the products meet the landmark indicators. At the same time, more detailed grading is often established within the enterprise, and Yuyue will make internal grade adjustments to different batches of soy sauces to ensure the consistent flavor of the final product. In the process of quality standardization, process control standards are also indispensable. The company will formulate quality control points and parameter ranges for the entire process from raw material acceptance to fermentation, sterilization, and packaging. The application of laboratory rapid
Molecular Microbiology Research, 2025, Vol.15, No.2, 45-58 http://microbescipublisher.com/index.php/mmr 53 analysis technology improves standardization efficiency. For example, near-infrared spectroscopy technology can determine the amino acid nitrogen and salt of soy sauce in a few minutes to achieve online monitoring. These technical means reduce the errors in artificial quality control. Standardization also involves the establishment of sensory evaluation standards. By training the sensory evaluation team, they were able to compare the standard samples and unify the flavor description language and scoring standards. For example, determine the definition and intensity grading of the "sauce flavor" attribute, as well as the corresponding reference samples (Imamura, 2016). Another aspect of quality standardization is production operation standardization. Traditional brewing relies on the master's experience in hand. In order to produce on a large scale, these hidden knowledge needs to be transformed into clear norms. These operating specifications combine real-time monitoring and recording to form standard operating procedures (SOPs) so that skills can be copied and inherited. During the inheritance process, Yuyue compiled detailed traditional craftsmanship rules, literalizing the oral experiences of masters of all generations, and providing textbooks for the training of the new generation of brewers. 6.3 Sensory quality and consumer preferences Quality evaluation ultimately depends on consumer acceptance. Different production processes and formulas may lead to differences in product flavors, and whether consumers buy it is an important indicator to judge whether the innovation is successful or not. Research and market research in recent years have shown that consumers have a tendency to prefer certain sensory attributes of soy sauce while meeting basic umami flavor and mellowness. Consumer testing pointed out that soy sauce with dark colors, high viscosity and obvious fresh sweetness are more popular among Korean consumers. This coincides with the characteristics of long-term fermented soy sauce and explains why brewed soy sauce (naturally fermented soy sauce) is often more popular than formulated soy sauce made with chemical hydrolysis. To quantify consumer preferences, preference mapping analysis can be used to correlate sensory attributes with consumer preferences. Through data analysis of large amounts of consumer ratings, we can find key attributes to drive preferences. Expert research combines sensory assessment and metagenomic data and proposes that sensory quality can be affected by regulating the fermentation microbiome: for example, increasing the proportion of Tetragenococcus helps increase umami flavor, while moderately retaining yeast brings aroma and softness. These findings provide new ideas for improving consumer sensory experience from a microbial perspective. 7 Food Safety Control and Risk Prevention and Control mechanism 7.1 Raw material screening and pollution source control Food safety is the bottom line that must be strictly adhered to in the modern transformation of traditional soy sauce brewing, and the first step is the raw material level. High-quality and safe raw materials are the basis for brewing safe and mellow soy sauce. When screening raw materials, you should ensure that the main ingredients such as soybeans and flour are free from mycotoxin contamination, no pesticide residues exceed the standard, and meet the food-grade microbial limit standards. Studies have shown that in fermented soybean foods, raw materials are often one of the main sources of introduction of harmful fungi and their toxins. If the control is not strictly controlled, toxic metabolites such as aflatoxin may enter the fermentation system with the raw materials. Aspergillus commonly used in soy sauce brewing must be of reliable origin to ensure that the strain used does not produce toxins. Brewing practice in Japan shows that through breeding and monitoring, only using Aspergillus, which does not produce aflatoxin, can effectively avoid the risk of introducing toxins in the pre-fermentation stage. In addition to mycotoxins, pathogenic bacteria contamination in raw materials also needs attention. If soybeans are contaminated by salmonella and E. coli during storage, they may reproduce in the early stages of sauce making and create safety hazards (Kim et al., 2023). Excipients such as water sources and salt must also meet food safety standards to prevent heavy metals or other harmful impurities from being mixed in. Pollution source control is not limited to the raw materials themselves, but also includes the processing environment and packaging materials. In the traditional open-air sauce drying process, birds, insects, etc. should prevent miscellaneous
Molecular Microbiology Research, 2025, Vol.15, No.2, 45-58 http://microbescipublisher.com/index.php/mmr 54 bacteria or foreign objects from falling into the fermentation sauce blank. Physical covering or regular deworming measures can be used. Implement good production specifications (GMP) in the entire production environment, keep the ground and container clean and free of mold spots, strictly partition the raw material area and fermentation area to avoid cross-contamination. 7.2 Health code management and key control point identification (HACCP) In the process of integrating traditional soy sauce brewing into modern industrial production, it is extremely important to establish a systematic health standard management and Critical Control Points (CCP) monitoring system. Hazard Analysis and Critical Control Points (HACCP) is a preventive food safety management system widely recognized internationally. Applying its principles to soy sauce production can effectively identify and control potential risks. It is necessary to formulate a sanitation standard operating procedure (SSOP) covering the entire production process, including equipment cleaning and disinfection, personnel hygiene, insect and rat prevention and environmental monitoring, etc., to create a clean fermentation environment. The fermentation workshop should conduct regular air and surface microbial testing, and key areas such as the koji room and the surrounding fermentation tank should be kept at a low bacterial load to prevent miscellaneous bacteria from invading the fermentation system. Through HACCP analysis, the koji fermentation stage, dew fermentation stage, finished product sterilization and packaging stage can be set as the CCP for key monitoring. During the bellowing stage, temperature and humidity control are key points, so constant bellowing conditions should be maintained through the automatic control system, and alarm and correct immediately once deviation is deviated. During the fermentation stage of sunburn, we need to pay attention to changes in salt concentration and pH, which is directly related to whether the obstacles to the inability to grow pathogens are sufficient. When it is detected that the salt concentration is below the safety threshold, salt supplementation or adjustment should be made in time to restore the antibacterial environment (Figure 3). Finished pasteurization is the last key control point, and it is necessary to verify that the heating temperature and time are sufficient to kill salt-resistant spores and pathogenic bacteria without excessive damage to the flavor of the soy sauce. In practice, traditional soy sauce brewing companies have achieved good results in implementing the HACCP system. Some companies have successfully passed ISO 22000 and HACCP certification, marking that their risk control in the entire process from raw materials to finished products meets international standards (Sun et al., 2010). Skowron's team pointed out that the lack of standardized hygiene control and safety standards will significantly increase the risk of pathogenic bacteria or toxins in fermented foods, which indirectly confirms the necessity of implementing systems such as HACCP. By training employees to identify hazardous factors in each link and strictly follow the regulations, the safety risks during soy sauce brewing will be greatly reduced. Figure 3 Microbial contamination control and environmental sanitation management in open fermentation systems 7.3 Application of microbial risk warning and rapid detection technology In view of the microbial risks in the soy sauce brewing process, establishing a fast and sensitive early warning and detection mechanism is one of the key measures to improve the level of food safety guarantee. During long-term
Molecular Microbiology Research, 2025, Vol.15, No.2, 45-58 http://microbescipublisher.com/index.php/mmr 55 natural fermentation, the bacteria that may breed and the toxins they produce need to be discovered and controlled in a timely manner. The development of modern food microbial detection technology provides a variety of means for this. Polymerase chain reaction (PCR) and its quantitative version (qPCR) have been widely used for rapid detection of pathogenic bacteria in food, and can identify whether there are common pathogenic bacteria such as Salmonella and Staphylococcus aureus in the fermentation broth within a few hours. Compared with the traditional culture method, molecular detection methods greatly shorten the detection cycle and achieve early warning of pathogenic microorganisms during fermentation. In addition to nucleic acid testing, rapid immunology detection technology also plays an important role in monitoring soy sauce toxins. Taking aflatoxin B₁ as an example, it used to require tedious sample pretreatment and chromatography analysis. Now some researchers have developed rapid test strips for immunochromatography-based soy sauce matrix. It is possible to determine whether the toxin in the sample exceeds the standard within a few minutes without extraction. Wang et al. (2016) reported a rapid immunochromatography method based on fluorescent microsphere labeling. The visible detection limit of aflatoxin B₁ in soy sauce is up to 2.5 μg/L, and has quantitative analysis capabilities. This rapid detection technology has shown good application prospects in the quality monitoring of fermented products such as soy sauce, and can be used as a powerful tool for daily batch inspection and risk investigation. 8 The Path of Integration Between Tradition and Innovation 8.1 The inheritance and industrialization of intangible cultural heritage of sauce-making "Fishing Soy Sauce Brewing Technique" is an intangible cultural heritage originated from the Northern Song Dynasty. Its protection and inheritance have important cultural value. In the modern context, achieving the sustainable inheritance of this traditional technique requires exploring the development path of the integration of culture and industry. On the one hand, the core elements of traditional brewing processes should be respected and retained, including the experience and skills contained in natural koji making, open-air fermentation and manual operation. This can ensure that the skills are passed down from generation to generation by establishing intangible cultural heritage skills inheritance bases, master-apprentice training, etc., and record and organize traditional process parameters. On the other hand, promoting intangible cultural heritage skills to a broader market is inseparable from the help of industrialization. After being included in the municipal and provincial intangible cultural heritage (ICH) lists, "Yuyue Soy Sauce" from Lishui, Zhejiang, has seen active collaboration between local governments and enterprises. Resources have been invested in brand development and production upgrades, enabling this traditional soy sauce to embark on the path toward scaled industrial production (Figure 4). This exemplifies the commercial transformation potential of traditional craftsmanship—when the cultural value-added is recognized, traditional products can command a market premium, thereby fostering a virtuous cycle of “industry sustaining heritage and heritage enhancing industry” (O’Toole, 2019). Figure 4 Visual presentation of soy sauce product label design and brand communication The industrialization of Yuyue soy sauce technology can be considered as a combination of "regional brand + cultural experience": while retaining the characteristics of hand-made brewing, modern factory management is introduced to expand production capacity, and industrial tourism, brewing experience, etc. are developed in the
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