Method for preparing rice enzyme by using lactobacillus mucilaginosus enhanced fermentation

Abstract

The invention discloses a method for preparing a rice enzyme through enhanced fermentation of lactobacillus mucilaginosus, and belongs to the technical field of microorganisms. The rice enzyme prepared by adopting the method disclosed by the invention is relatively high in protein content, rich in various amino acids, B vitamins and organic acids, higher in nutritional value than a purely blended sour soup base, and capable of stimulating appetite and tonifying spleen, and the organic acids generated by fermentation can stimulate saliva and gastric acid secretion, enhance appetite and contribute to digestion, and the attention is gradually increased. The special fermentation inoculant and process parameters suitable for the rice enzyme process are obtained, the fermentation efficiency is greatly improved, the flavor and safety are improved, the loss is reduced, the production cost is reduced, and basic theories and data references are provided for standardized production of rice enzymes.

Description

Technical Field

[0001] This invention relates to a method for preparing rice enzyme by enhancing fermentation using fermented Lactobacillus mucilaginosus, belonging to the field of microbial technology. Background Technology

[0002] Rice enzymes have a high protein content and are rich in various amino acids, B vitamins, and organic acids, making them more nutritious than simply seasoned sour soup bases. They can also stimulate appetite and improve digestion; the organic acids produced during fermentation can stimulate saliva and gastric acid secretion, enhancing appetite and aiding digestion, leading to increasing interest. However, most rice enzyme production currently relies on traditional natural fermentation, which suffers from long production cycles, high costs, contamination by other microorganisms, lack of standardization, and unstable quality. The source of the fermented grains depends on local distilleries, resulting in significant variations in raw materials, brewing processes, grain freshness, and microbial communities, leading to large fluctuations in the "base flavor" of the rice enzymes. The fermentation process is uncontrollable, occurring naturally and relying primarily on microorganisms (lactic acid bacteria, acetic acid bacteria, yeast, etc.) in the air or attached to containers. The fermentation cycle, acidity, and the amount of flavor compounds produced are difficult to control precisely, making them susceptible to contamination by other microorganisms. Manual operation results in low production capacity, unable to meet large-scale market demand. Furthermore, the highly open production environment makes it difficult to control food safety risks (such as mold contamination, foreign objects, and excessive microorganisms).

[0003] Therefore, the purpose of this invention is to obtain a special fermentation agent and process parameters suitable for rice enzyme production, which can significantly improve fermentation efficiency, enhance flavor and safety, reduce losses and lower production costs, and provide basic theory and data reference for the standardized production of rice enzyme. Summary of the Invention

[0004] This invention provides a fermentation method for Lactobacillus mucilaginosus ( Limosilactobacillus fermentum R1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 26, 2025, with accession number GDMCC No: 67362.

[0005] The present invention also provides a composition containing the above-mentioned fermenting Lactobacillus mucinus R1.

[0006] In one embodiment of the present invention, the composition comprises one or more of the following: live strain of *Lactobacillus fermentum* R1, dry strain of *Lactobacillus fermentum* R1, metabolites of *Lactobacillus fermentum* R1, and inactivated strain of *Lactobacillus fermentum* R1.

[0007] In one embodiment of the present invention, the composition contains at least 1 × 10⁻⁶ Lactobacillus fermentans R1. 8 CFU / mL or 1×10 8 CFU / g.

[0008] The present invention also provides a microbial agent, wherein the microbial agent contains the above-mentioned fermenting Lactobacillus mucinus R1; In one embodiment of the present invention, the content of *Lactobacillus fermentans* R1 in the microbial agent is at least 1 × 10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0009] The present invention also provides a product containing the above-mentioned fermented Lactobacillus mucinus R1, or containing the above-mentioned composition or the above-mentioned microbial agent.

[0010] In one embodiment of the present invention, the product is food, medicine, or health product.

[0011] In one embodiment of the present invention, the food includes solid food, liquid food, semi-solid food, or fermented food.

[0012] In one embodiment of the present invention, the fermented food includes any one of dairy products, soy products, and fruit and vegetable products.

[0013] In one embodiment of the present invention, the dairy product includes any one of milk, sour cream, and cheese.

[0014] In one embodiment of the present invention, the fruit and vegetable products include any one of cucumber, carrot, beet, celery, and cabbage products.

[0015] The present invention also provides the application of the above-mentioned fermented Lactobacillus mucinus R1 or the above-mentioned composition or the above-mentioned microbial agent in the preparation of fermented foods.

[0016] In one embodiment of the present invention, the fermented product is a fermented alcoholic beverage or fermented vinegar.

[0017] In one embodiment of the present invention, the fermented alcoholic beverage is fruit wine, and the fermented vinegar is fruit vinegar; In one embodiment of the present invention, the fermented alcoholic beverage is yellow wine, cooking wine, rice wine, sweet rice wine, wine, beer, or baijiu; and the fermented vinegar is edible vinegar.

[0018] The present invention also provides the application of the above-mentioned fermented Lactobacillus mucin R1 or the above-mentioned composition or the above-mentioned microbial agent in the preparation of rice enzyme.

[0019] In one embodiment of the present invention, the rice enzyme can be used to flavor soup base.

[0020] This invention provides a method for preparing rice enzyme, wherein the method involves adding fermenting Lactobacillus mucilaginosus R1 or the aforementioned composition or the aforementioned microbial agent during the rice enzyme preparation process to carry out fermentation to prepare rice enzyme.

[0021] In one embodiment of the present invention, the method specifically includes: placing 4-degree distiller's grains at a material-to-water ratio of 1:1~2 (m / v, g / ml) in a pulping machine for pulping, followed by sterilization to obtain sterilized distiller's grains slurry; inoculating the sterilized distiller's grains slurry with *Lactobacillus fermentatus* R1, culturing at 35~38℃ for 12~13 h, then transferring to new sterilized distiller's grains slurry, culturing at 35~38℃ for 6~7 h, until the bacterial concentration reaches 10. 8 CFU / mL was used to obtain the bacterial agent; The prepared microbial agent was inoculated into 4°C distiller's grains (no sterilization required). After inoculation, the mixture was placed in a fermentation tank, sealed with gauze, and fermented at 28°C–35°C for 20–28 hours. Preferably, the concentration of the microbial agent was 1 × 10⁻⁶. 8 ~1.5×10 8 CFU / mL.

[0022] In one embodiment of the present invention, the method involves placing 4-degree distiller's grains in a pulping machine at a material-to-water ratio of 1:1.6 (m / v, g / ml) and pulping them.

[0023] The prepared microbial agent was inoculated into 4-degree distiller's grains (no sterilization required) at a 1% (w / w) inoculation rate, placed in a fermentation tank, sealed with gauze to ensure good air permeability, and placed in a 30°C constant temperature incubator for fermentation for 24 hours (until the acidity reaches 0.6 degrees).

[0024] In one embodiment of the present invention, the preparation method of the 4-degree distiller's grains is as follows: Wash the white glutinous rice and steam it for 40-50 minutes after the water boils. After stopping the steaming, cover the rice and let it sit for 5-10 minutes. Spread the steamed glutinous rice out and let it cool to 25-30°C. Add yeast to the cooled glutinous rice, with the amount of yeast added being 0.5-0.6% of the weight of the raw rice. Stir after adding the yeast. Place the glutinous rice mixed with yeast in a fermentation tank, seal it with plastic wrap to ensure good sealing, and place it in a constant temperature incubator at 28-30°C for fermentation for 40-45 hours.

[0025] Beneficial effects (1) Compared with natural fermentation, inoculation with Lactobacillus mucilaginosus R1 significantly shortened the fermentation time, and the rice enzyme product had a better color and appearance, a richer aroma, a more delicate taste, milder acidity, better flavor, no irritation, and higher overall sensory acceptance. Compared with natural fermentation, the amount of sweet amino acids increased significantly, enhancing the flavor layers of the sample without masking the main flavor. In addition, the cystine (cys-s) content doubled during the intensified fermentation process. Cystine is a sulfur-containing amino acid and is a key precursor for the generation of various high-grade flavor substances in microbial fermentation and chemical reactions. During brewing and storage, it undergoes Maillard reaction, producing a large amount of melanoidins and other substances, which are powerful natural antioxidants. This usually indicates that the sample is of higher quality, more fully brewed, and has a more complex flavor.

[0026] (2) The rice enzyme obtained by inoculating *Lactobacillus fermentans* R1 not only increases the organic acid content, giving the product a natural fermented sour taste and enhancing the flavor profile, but also improves the "freshness," masks some bitterness and fishiness, and has good synergy with umami. It also produces a small amount of fumaric acid, which quickly imparts a refreshing sour taste to the product, enhancing the flavor profile and masking some off-flavors. The enhanced fermentation process significantly reduces the content of acetic acid and ethyl acetate, making the sourness more mellow. In particular, excessive ethyl acetate combined with acetic acid can produce an unripe, green banana-like astringent taste, which may even be mistaken for a "rotten" or "spoiled" odor. Simultaneously, the enhanced fermentation process produces various flavor compounds such as isoamyl alcohol, 1-hexanol, 2-nonanone, 1-nonanol, and phenethyl acetate, making the product's aroma more mellow and long-lasting. Notably, it produces special flavor compounds such as acetoin, propylene glycol, and ethyl 9-decenoate, adding a slight milky and fruity sweetness.

[0027] Preservation of biological materials Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum R1, categorized as R1 Limosilactobacillus fermentum It was deposited on November 26, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67362. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0028] Figure 1 High salt tolerance of fermenting Lactobacillus mucinus.

[0029] Figure 2 The tolerance of fermenting Lactobacillus mucilaginosus to phenolic substances.

[0030] Figure 3 Acetic acid tolerance of fermenting Lactobacillus mucilaginosus.

[0031] Figure 4 : The ethanol tolerance of fermenting Lactobacillus mucilaginosus.

[0032] Figure 5 : The acetaldehyde tolerance of fermenting Lactobacillus mucilaginosus.

[0033] Figure 6 : The ethanol tolerance of fermenting Lactobacillus mucilaginosus.

[0034] Figure 7 Hydrogen peroxide tolerance of fermenting *Lactobacillus mucinus*.

[0035] Figure 8 Temperature tolerance of fermenting *Lactobacillus mucinus*.

[0036] Figure 9 The tolerance of fermenting *Lactobacillus mucilaginosus* to inorganic salts.

[0037] Figure 10 The ability of fermenting Lactobacillus mucilaginosus to tolerate high sugar levels.

[0038] Figure 11 pH tolerance of fermenting Lactobacillus mucinus.

[0039] Figure 12 The bile salt tolerance of fermenting *Lactobacillus mucilaginosus*.

[0040] Figure 13 : Carbon source utilization ability of fermenting Lactobacillus mucilaginosus.

[0041] Figure 14 Sensory evaluation of fermented Lactobacillus mucilaginosus. Detailed Implementation

[0042] Unless otherwise specified, the technical means used in the following embodiments are conventional technical means well known to those skilled in the art. Unless otherwise specified, the reagents used in the embodiments are commercially available.

[0043] Example 1: Screening of fermenting Lactobacillus mucinus Take 0.1 mL of the stock solution of 10 commercially available rice enzyme products, spread it on MRS agar medium, and take 3 replicates. Incubate at 37℃ for 48 h. Take 1 mL of the rice enzyme product stock solution with colony growth and add it to 9 mL of sterile physiological saline. Mix well to prepare 10 -1 10 -2 10 -3 10 -4Serial dilutions were prepared. 0.1 mL of each serial dilution was spread onto MRS agar medium, with three replicates, and incubated at 37 ℃ for 48 h. The morphology of colonies on the plates was observed. Single colonies of varying sizes, shapes, and colors that showed good growth were selected and streaked onto MRS agar medium using the streak plate method, incubated at 37 ℃ for 48 h. After three generations of streak purification, the culture was transferred to test tube slants, numbered, and incubated for 48 h. The culture was then stored at 4 ℃ and prepared into glycerol tubes for preservation at -80 ℃.

[0044] The lactic acid bacteria obtained through screening were sequenced and identified as... Limosilactobacillus fermentum (Lactobacillus fermentans). It was sent to a preservation center for preservation and named: Lactobacillus fermentans R1.

[0045] Example 2: Performance of Fermenting Lactobacillus strain When testing the performance of the following Lactobacillus fermentans strains, the initial inoculum size was 1% (v / v). 1. High salt tolerance: (1) Prepare MRS liquid culture media with final NaCl concentrations (w / w) of 2%, 4%, 6%, 8%, 10%, and 15%, respectively; (2) The fermenting *Lactobacillus mucinus* R1 obtained in Example 1 was inoculated into the liquid culture medium obtained in step (1), and after being cultured in a constant temperature incubator at 37°C for 24 hours, the OD of the fermenting *Lactobacillus mucinus* was detected. 600 Meanwhile, the culture was carried out in MRS liquid medium without added (0%) NaCl as a control.

[0046] The results show that ( Figure 1 ): It was found that the growth of fermenting Lactobacillus was significantly inhibited as the NaCl concentration increased. After culturing in MRS liquid medium containing 8% NaCl for 24 h, the bacterial cell concentration increased, but growth was still possible. When the NaCl content reaches 10% and 15%, the strain hardly grows. As the osmotic pressure increases, the inhibitory effect on the growth of fermenting *Lactobacillus mucilaginosus* becomes more pronounced.

[0047] These results indicate that the strain has a certain tolerance to high salt concentrations, able to tolerate 8% salt concentration, and still has a 20% survival rate at 10% salt concentration.

[0048] 2. Tolerance to phenolic substances: (1) Prepare MRS liquid culture media with final phenol concentrations (w / w) of 0.05%, 0.1%, 0.2%, 0.3%, and 0.4%, respectively; (2) The fermenting *Lactobacillus mucinus* R1 obtained in Example 1 was inoculated into the liquid culture medium obtained in step (1), and after being cultured in a constant temperature incubator at 37°C for 24 hours, the OD of the fermenting *Lactobacillus mucinus* was detected. 600 Meanwhile, the culture was carried out in MRS liquid medium without added (0%) phenol as a control.

[0049] The results show that ( Figure 2 ): It was found that the growth inhibition of *Lactobacillus fermentans* became more significant with increasing phenol concentration. After 24 hours of culture in MRS liquid medium containing 0.2% phenol, the strain could still grow even with increased cell concentration; however, when the phenol concentration reached 0.3%, the strain showed almost no growth. The inhibitory effect of phenol on the growth of *Lactobacillus fermentans* became increasingly pronounced with increasing phenol concentration.

[0050] These results indicate that the strain has a certain tolerance to phenolic substances, can tolerate a phenol concentration of 0.2%, and still has a 10% survival rate at a phenol concentration of 0.3%.

[0051] 3. Tolerance to dual stress of ethanol and acetic acid: (1) When the amount of ethanol added is constant at 6% (w / w), the OD of fermenting Lactobacillus mucilaginosus was measured after culturing in MRS medium containing different concentrations of acetic acid for 24 h. 600 The details are as follows: 1) Prepare MRS liquid culture media with an ethanol concentration of 6% and final acetic acid concentrations (w / w) of 0.2%, 0.4%, 0.6%, and 0.8%, respectively; 2) The fermenting *Lactobacillus mucinus* R1 obtained in Example 1 was inoculated into the liquid culture medium obtained in step (1), and after being cultured in a constant temperature incubator at 37°C for 24 hours, the OD of the fermenting *Lactobacillus mucinus* was detected. 600 Meanwhile, the culture was carried out in MRS liquid medium with only 6% ethanol and no acetic acid (0%) as a control.

[0052] The results show that ( Figure 3 ): It was found that the growth of *Lactobacillus fermentans* was significantly inhibited as the acetic acid concentration increased. After culturing in MRS liquid medium containing 0.6% acetic acid for 24 h, the bacterial cell concentration increased and growth was still possible; however, when the acetic acid concentration reached 0.8%, the strain hardly grew.

[0053] (2) When the acetic acid concentration remained constant at 0.4% (w / w), the OD of fermenting *Lactobacillus mucinus* was measured after culturing in MRS medium containing different concentrations of ethanol for 24 h. 600 The details are as follows: 1) Prepare MRS liquid culture media with acetic acid concentration of 0.4% and final ethanol concentration (w / w) of 3%, 6%, 9%, and 12%, respectively; 2) The fermenting *Lactobacillus mucinus* R1 obtained in Example 1 was inoculated into the liquid culture medium obtained in step (1), and after being cultured in a constant temperature incubator at 37°C for 24 hours, the OD of the fermenting *Lactobacillus mucinus* was detected. 600 Meanwhile, the culture was carried out in MRS liquid medium with only 0.4% acetic acid and no ethanol (0%) as a control.

[0054] The results show that ( Figure 4 ): It was found that the growth of fermenting Lactobacillus was significantly inhibited as the ethanol concentration increased. After culturing in MRS liquid medium containing 9% ethanol for 24 h, the bacterial cell concentration increased and the strain could still grow; however, when the ethanol concentration reached 12%, the strain hardly grew.

[0055] These results indicate that the strain has good tolerance to dual stress of ethanol and acetic acid.

[0056] 4. Acetaldehyde tolerance: The OD of *Lactobacillus fermentans* cultured for 24 h in MRS medium containing different concentrations of acetaldehyde was measured. 600 The specific steps are as follows: (1) Prepare MRS liquid culture media with final acetaldehyde concentrations of 0.1%, 0.2%, 0.3%, and 0.5%, respectively; (2) The fermenting *Lactobacillus mucinus* R1 obtained in Example 1 was inoculated into the liquid culture medium obtained in step (1), and after being cultured in a constant temperature incubator at 37°C for 24 hours, the OD of the fermenting *Lactobacillus mucinus* was detected. 600 Meanwhile, the culture was carried out in MRS liquid medium without added (0%) acetaldehyde as a control.

[0057] The results show that ( Figure 5 ): It was found that the growth of *Lactobacillus fermentans* was significantly inhibited with increasing acetaldehyde concentration. After 24 hours of culture in MRS liquid medium containing 0.2% acetaldehyde, the bacterial cell concentration increased, yet growth continued; however, when the acetaldehyde concentration reached 0.3%, the strain showed almost no growth. The inhibitory effect of acetaldehyde on the growth of *Lactobacillus fermentans* became increasingly pronounced with increasing acetaldehyde concentration.

[0058] These results indicate that the strain has a certain tolerance to acetaldehyde, being able to tolerate 0.2% acetaldehyde and still maintaining a 10% survival rate at a 0.3% acetaldehyde concentration.

[0059] 5. Ethanol tolerance: The OD of *Lactobacillus fermentans* cultured for 24 h in MRS medium containing different concentrations of ethanol was measured. 600 The specific steps are as follows: (1) Prepare MRS liquid culture media with final ethanol concentrations (w / w) of 3%, 6%, 9%, 12%, and 15%, respectively; (2) The fermenting *Lactobacillus mucinus* R1 obtained in Example 1 was inoculated into the liquid culture medium obtained in step (1), and after being cultured in a constant temperature incubator at 37°C for 24 hours, the OD of the fermenting *Lactobacillus mucinus* was detected. 600 Meanwhile, the culture was carried out in MRS liquid medium without added (0%) ethanol as a control.

[0060] The results show that ( Figure 6 ): It was found that the growth of *Lactobacillus fermentatus* was significantly inhibited with increasing ethanol concentration. After 24 hours of culture in MRS liquid medium containing 9% phenol, the bacterial cell concentration increased, yet growth continued; however, when the ethanol concentration reached 12%, the strain showed almost no growth. The inhibitory effect of ethanol on the growth of *Lactobacillus fermentatus* became increasingly pronounced with increasing ethanol concentration.

[0061] These results indicate that the strain has a certain tolerance to ethanol, and can tolerate 9% ethanol concentration, while still maintaining a 15% survival rate at 12% acetaldehyde concentration.

[0062] 6. Tolerance to H2O2: The OD of fermenting *Lactobacillus mucinus* was determined by measuring the OD values ​​of *Lactobacillus mucinus* cultured in MRS medium containing different concentrations of H2O2 for 24 h. 600 The specific steps are as follows: (1) Prepare MRS liquid culture medium with final H2O2 concentrations of 1 mM, 2 mM, 3 mM and 5 mM respectively; (2) The fermenting *Lactobacillus mucinus* R1 obtained in Example 1 was inoculated into the liquid culture medium obtained in step (1), and after being cultured in a constant temperature incubator at 37°C for 24 hours, the OD of the fermenting *Lactobacillus mucinus* was detected. 600 Meanwhile, the culture was carried out in MRS liquid medium without added (0%) hydrogen peroxide as a control.

[0063] The results show that ( Figure 7 ): It was found that the growth of *Lactobacillus fermentans* was significantly inhibited with increasing H2O2 concentration. After 24 h of culture in MRS liquid medium containing 2 mM H2O2, the bacterial cell concentration increased, yet growth continued; however, when the H2O2 concentration reached 3 mM, the strain showed almost no growth. The inhibitory effect of H2O2 on the growth of *Lactobacillus fermentans* became increasingly pronounced with increasing H2O2 concentration.

[0064] These results indicate that the strain has a certain degree of tolerance to H2O2. It can tolerate 2 mM H2O2 and still has a 10% survival rate at 3 mM H2O2.

[0065] 7. Temperature tolerance: Different temperatures may affect the fermentation efficiency of *Lactobacillus mucinus*. By setting multiple temperature gradients, the OD (oxidative stress) of *Lactobacillus mucinus* after culturing at different temperatures for 24 h was measured. 600 It was found that fermented *Lactobacillus mucinus* had an OD of 20°C at a temperature of 20°C. 600 The concentration is around 0.3, indicating slow growth. Growth is faster and less differentiated at temperatures between 30-42℃. However, growth is significantly inhibited at 50℃, suggesting that the optimal temperature range for *Lactobacillus fermentatus* is 30-42℃; excessively high or low temperatures are unsuitable for its growth. Figure 8 ).

[0066] 8. Tolerance to inorganic salts: A general or specific deficiency of inorganic salts can cause multidimensional growth inhibition and physiological damage to *Lactobacillus fermentans*. By setting up specific deficiencies of different types of inorganic salts, the OD (oxidative stress) of *Lactobacillus fermentans* after 24 h of culture was measured. 600 The study found that the growth of *Lactobacillus fermentatus* was significantly inhibited, indicating that inorganic salts are crucial for the growth of *Lactobacillus fermentatus*. Figure 9 ).

[0067] 9. High sugar tolerance: The OD of *Lactobacillus fermentans* cultured for 24 h in MRS medium containing different concentrations of glucose was measured. 600 To assess the strain's tolerance to high sugar, the specific steps are as follows: (1) Prepare MRS liquid culture media with final glucose concentrations (w / w) of 0.2%, 0.5%, 1%, 5%, 10%, and 20%, respectively; (2) The fermenting *Lactobacillus mucinus* R1 obtained in Example 1 was inoculated into the liquid culture medium obtained in step (1), and after being cultured in a constant temperature incubator at 37°C for 24 hours, the OD of the fermenting *Lactobacillus mucinus* was detected. 600 Meanwhile, the culture was carried out in MRS liquid medium without added (0%) hydrogen peroxide as a control.

[0068] The results show that ( Figure 10 ): When glucose concentration is <0.5%, OD is found to be low. 600At a glucose concentration of around 0.3, the growth is slow. At a glucose concentration of 0.5-5%, the growth is faster. When the glucose concentration reaches 10%, the growth of the strain begins to be inhibited. When the glucose concentration reaches 20%, the growth is basically stopped.

[0069] The results showed that the strain had a certain tolerance to high sugar levels. It could tolerate 10% glucose and still had a 20% survival rate at a 20% glucose concentration.

[0070] 10. pH tolerance: The OD of fermenting *Lactobacillus mucinus* was measured after culturing in MRS media with different pH values ​​for 24 h. 600 It was found that the strain barely grew at pH < 4, grew rapidly between pH 5 and 7, and its growth began to be inhibited at pH 8, and almost stopped at pH 9. This indicates that the optimal pH range for *Lactobacillus fermentans* is between 5 and 7; excessively high or low pH levels are unsuitable for its growth. Figure 11 ).

[0071] 11. Tolerance to bile salts Lactic acid bacteria in the human body mainly colonize the intestines. Digestive juices are primarily composed of bile and pancreatic juice. The concentration of bile salts in the human small intestine is typically maintained within the range of 0.05%-0.30%. In the presence of bile salts, lactic acid bacteria can maintain a high survival rate, which is a crucial basis for their probiotic effects. The OD of this strain after 24 hours of incubation under different concentrations of bile salts was measured. 600 The specific steps for assessing the strain's tolerance to bile salts are as follows: (1) Prepare MRS liquid culture media with final bile salt concentrations (w / w) of 0.05%, 0.1%, 0.2%, and 0.4%, respectively; (2) The fermenting *Lactobacillus mucinus* R1 obtained in Example 1 was inoculated into the liquid culture medium obtained in step (1), and after being cultured in a constant temperature incubator at 37°C for 24 hours, the OD of the fermenting *Lactobacillus mucinus* was detected. 600 Meanwhile, the culture was carried out in MRS liquid medium without added (0%) bile salts as a control.

[0072] The results show that ( Figure 12 ): The strain exhibited excellent growth at a bile salt concentration of 0.05%, almost identical to the control; however, when the bile salt concentration exceeded 0.10%, OD... 600 The rapid decline suggests that this strain has a certain degree of tolerance to bile salts.

[0073] 12. Ability to utilize carbon sources By measuring the OD600 of this strain after culturing it with different concentrations of starch for 24 h, it was found that this strain cannot utilize starch through its own metabolic hydrolysis. Such strains tend to use small molecule sugars such as glucose and maltose as carbon sources to maintain growth and metabolism.

[0074] Example 3: Production of Rice Enzyme I. Rice Enzyme Production Process 1. Production process of 4-degree distiller's grains (1) Rice selection: Select high-quality white glutinous rice and remove impurities and grains.

[0075] (2) Washing rice: Rinse 1-2 times, and try to avoid stirring too much.

[0076] (3) Steaming rice: After washing the white glutinous rice, rinse it with water to remove the rice milk and then steam it. The steaming time is generally 40 minutes after the water boils. Steam until the glutinous rice grains are hard on the outside and soft on the inside, loose and not sticky, plump, and without white core. After stopping the steaming, cover the rice and let it sit for 5 minutes.

[0077] (4) Cooling: Spread out the steamed glutinous rice and let it cool to about 30°C. The faster this operation is performed, the better. Try to avoid exposing the steamed rice to the air for too long, which could lead to contamination by other bacteria.

[0078] (5) Adding yeast: The amount of yeast (Yada brand yeast, purchased from Taobao e-commerce platform) added is 0.5% of the weight of raw rice. Stir after adding yeast.

[0079] (6) Fermentation: Seal the fermentation tank with the mixed koji with plastic wrap to ensure good sealing, and place it in a 28℃ constant temperature incubator for fermentation for 40 h.

[0080] 2. Preparation of rice enzyme (1) Preparation of rice enzyme by fermenting Lactobacillus mucilaginosus R1 1) Place the 4-degree distiller's grains obtained in step 1 into a pulper at a material-to-water ratio of 1:1.6 (m / v, g / ml) for pulping (pulping time is 5 min). After pulping, sterilize at 121℃ for 15 min. After the sterilization, place it in a clean bench to cool and wait for use to obtain sterile distiller's grains pulp.

[0081] 2) Remove the slant test tube containing the bacterial culture (Lactobacillus fermentata R1) from the 4°C freezer. Use a 10 µL inoculation loop to scrape a loopful of the culture from the surface into a test tube containing 5 mL of sterile distiller's grains. Incubate at 37°C for 12 h, then transfer to an Erlenmeyer flask containing 45 mL of sterile distiller's grains and incubate at 37°C for 6 h, until the bacterial concentration reaches 10⁻⁶. 8 The inoculum can be obtained by using CFU / mL.

[0082] Inoculate the 4-degree lees obtained in step 1 with the inoculum at a mass fraction of 1% (w / w), place it in a fermentation tank, seal it with gauze to ensure good air permeability, and place it in a 30°C constant temperature incubator for fermentation for 24 hours (until the acidity reaches 0.6 degrees).

[0083] (2) Preparation of rice enzyme by fermenting Lactobacillus mucin CICC 24232 1) Place the 4-degree distiller's grains obtained in step 1 into a pulper at a material-to-water ratio of 1:1.6 (m / v, g / ml) for pulping (pulping time is 5 min). After pulping, sterilize at 121℃ for 15 min. After the sterilization, place it in a clean bench to cool and wait for use to obtain sterile distiller's grains pulp.

[0084] 2) Remove the slant test tube containing the bacterial culture (Lactobacillus fermentum CICC 24232) from the 4°C freezer. Use a 10 µL inoculation loop to scrape a loopful of the culture from the surface into a test tube containing 5 mL of sterile distiller's grains. Incubate at 37°C for 12 h, then transfer to an Erlenmeyer flask containing 45 mL of sterile distiller's grains and incubate at 37°C for 6 h, until the bacterial concentration reaches 10⁻⁶. 8 The inoculum can be obtained by using CFU / mL.

[0085] Inoculate the 4-degree lees obtained in step 1 with the inoculum at a mass fraction of 1% (w / w), place it in a fermentation tank, seal it with gauze to ensure good air permeability, and place it in a 30°C constant temperature incubator for fermentation for 28 hours (until the acidity reaches 0.6 degrees).

[0086] (3) Preparation of rice enzyme without adding microbial agents (natural fermentation group) The lees obtained in step 1 were placed in a pulping machine at a material-to-water ratio of 1:1.6 (m / v, g / ml) and pulped for 5 minutes. After stirring evenly, the mixture was placed in a fermentation tank and sealed with gauze to ensure good air permeability. The tank was then placed in a 30°C constant temperature incubator for fermentation for 48 hours (until the acidity reached 0.6 degrees).

[0087] II. Effects 1. Evaluation of physicochemical indicators: When the acidity reached 0.6 degrees, the natural fermentation time was 48 hours, the enhanced fermentation time using *Lactobacillus mucinus* CICC 24232 was 28 hours, and the enhanced fermentation time using *Lactobacillus mucinus* R1 was 24 hours. From the fermentation results, the enhanced fermentation using *Lactobacillus mucinus* R1 significantly shortened the fermentation time, had a slightly higher soluble sugar content than the other two groups, and the pH was basically the same. The results are shown in Table 1.

[0088] Table 1: Physicochemical Indicators of Different Groups

[0089] 2. Sensory rating When the fermentation acidity reached 0.6 degrees, compared with the naturally fermented group and the fermented *Lactobacillus mucinus* CICC 24232 group, the sample fermented with *Lactobacillus mucinus* R1 enhanced fermentation showed better color and appearance, a richer aroma, a more delicate taste, milder acidity, better flavor, no irritation, higher overall acceptability, and a higher comprehensive score. The results are as follows: Figure 14 As shown.

[0090] 3. Flavor Data Evaluation (1) The content of free amino acids (FAAs) is shown in Table 2.

[0091] Table 2: Free amino acid (FAAs) content (mg / 100g) in different groups

[0092] The results show: The naturally fermented sample contained 4.85±0.20 mg / 100g of sweet amino acids, while the sample fermented with *Lactobacillus mucilaginosus* R1 contained 5.40±0.20 mg / 100g. Compared to naturally fermented samples, the sweet amino acid content was significantly increased, enhancing the flavor profile without masking the main flavor. Furthermore, the cystine (cys-s) content doubled during the enhanced fermentation process. Cystine, a sulfur-containing amino acid, is a key precursor in the formation of various advanced flavor compounds during microbial fermentation and chemical reactions. It undergoes the Maillard reaction during brewing and storage, producing large amounts of melanoidins and other substances, acting as a powerful natural antioxidant. This typically indicates higher quality, more complete brewing, and more complex flavor profiles.

[0093] (2) Detection of organic acids Organic acids significantly influence the flavor and quality of products by regulating acidity, balancing sweetness, enhancing aroma, improving texture, and extending shelf life. Table 3 shows the content of the three main organic acids in rice enzymes, with acetic acid having the highest content, followed by lactic acid.

[0094] Table 3: Organic acid content in different groups (mg / 100g)

[0095] - indicates that it was not detected.

[0096] The results show: Enhanced fermentation increases the organic acid content, giving the product a natural fermented sour taste, enhancing its flavor profile, and improving its freshness. It also masks some bitterness and fishiness, exhibiting good synergy with umami. During fermentation, the acetic acid content increases, corresponding to a faster fermentation rate and the production of a small amount of fumaric acid. This quickly imparts a refreshing sour taste to the product, enhancing its flavor profile and masking some off-flavors.

[0097] (3) Detection of volatile flavor compounds A variety of volatile flavor compounds (VFCs), including alcohols, esters, and acids, were detected in rice enzymes, as shown in Table 4.

[0098] Table 4: Detection of volatile flavor compounds in different groups (μg / kg)

[0099] - indicates that it was not detected.

[0100] The content of ethyl acetate is 490.97 μg / kg. Experimental verification shows that when the content of ethyl acetate is higher than 1000 μg / kg, its flavor begins to show a slight solvent background, suppressing other delicate aromas and making it appear less natural and lacking in layers. When it is lower than 100 μg / kg, its flavor is almost imperceptible, and when mixed with other aromas, it contributes a very slight "freshness". Therefore, the content of ethyl acetate should be within the range of 100~1000 μg / kg. The acetic acid content is 1,248.76 μg / kg. Experimental verification shows that when the acetic acid content is higher than 3000 μg / kg, its flavor begins to have a pungent taste and presents a strong vinegar flavor. When it is lower than 1000 μg / kg, its flavor is a weak and adjustable sour taste. Therefore, the acetic acid content should be within the range of 1000~3000 μg / kg.

[0101] The results show: (1) Among them, esters are the main volatile flavor substances, accounting for about 70%, which endow the product with a variety of flavors such as fruity aroma, sweet aroma, and milky aroma. Ethyl acetate is the most important volatile ester flavor substance in the sample. Its relative content is slightly higher than that of other ester compounds. It has a strong fruity aroma and sweet aroma, similar to the aroma of pineapple, banana or pear. It is the key substance constituting the typical flavor characteristics of rice enzyme. In terms of sensory characteristics, it has the characteristic of high aroma intensity. Even a low content can improve the flavor of food. The synergistic effect of multiple volatile flavor substances enriches the overall flavor profile of rice enzyme and enhances its aroma layering, making it present a unique flavor characteristic.

[0102] (2) The enhanced fermentation process significantly reduced the content of acetic acid and ethyl acetate, making the acidity milder. In particular, excessive ethyl acetate combined with acetic acid can produce an unripe, green banana-like astringent taste, which may even be mistaken for a "rotten" or "spoiled" smell. At the same time, the enhanced fermentation process produced a variety of flavor substances such as isoamyl alcohol, 1-hexanol, 2-nonanone, 1-nonanol, and phenethyl acetate, making the aroma of the product more mellow and lasting. In particular, it produced special flavor substances such as acetoin, propylene glycol, and ethyl 9-decenoate, adding a slight milky and fruity sweetness.

[0103] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum R1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 26, 2025, with accession number GDMCC No: 67362.

2. A composition comprising the Limosilactobacillus fermentum R1 of claim 1.

3. The composition of claim 2, wherein, one or more of the following: a live strain of Limosilactobacillus fermentum R1, a dry strain of Limosilactobacillus fermentum R1, a metabolite of Limosilactobacillus fermentum R1, an inactivated strain of Limosilactobacillus fermentum R1; Preferably, the content of L. muci dos R1 in the composition is at least 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g.

4. A microbial inoculant, characterized in that, The microbial agent contains the Limosilactobacillus fermentum R1 of claim 1. Preferably, the content of L. fermentum R1 in the microbial inoculant is at least 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g.

5. A product characterized by, The product contains the Limosilactobacillus fermentum R1 of claim 1, or contains the composition of claim 2 or 3, or contains the microbial agent of claim 4, and the product is a fermented product, a food, a medicine or a health product; Preferably, the food includes a solid food, a liquid food, a semi-solid food or a fermented food; Preferably, the fermented food includes any one of dairy products, bean products or fruit and vegetable products; Preferably, the dairy products include any one of milk, sour cream or cheese; Preferably, the fruit and vegetable products include any one of cucumber, carrot, beet, celery or Chinese cabbage products; Preferably, the fermented product is rice ferment.

6. Use of the Limosilactobacillus fermentum R1 of claim 1 or the composition of claim 2 or 3 or the microbial agent of claim 4 in the preparation of a fermented product.

7. Use according to claim 6, characterized in that, The fermented product is a fermented alcoholic beverage, a fermented vinegar or a rice ferment.

8. Use according to claim 7, characterized in that, The fermented alcoholic beverage is fruit wine, and the fermented vinegar is fruit vinegar; Preferably, the fermented alcoholic beverage is yellow rice wine, cooking wine, rice wine, sweet rice wine, grape wine, beer or baijiu (Chinese liquor), and the fermented vinegar is edible vinegar.

9. A method of preparing rice ferment, characterized by, The method specifically includes:

10. The method of claim 9, wherein, After 4-degree distiller's grains are slurried and sterilized at a ratio of 1:1-2, the Limosilactobacillus fermentum R1 is inoculated and cultured to prepare a microbial agent; the prepared microbial agent is inoculated into 4-degree distiller's grains, and after inoculation, the fermenter is sealed with gauze and fermented at 28-35°C for 20-28 h. ​ Preferably, the concentration of the bacterial agent is 1 x 10 8 ~1.5 x 10 8 CFU / mL.

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