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
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