A lactic acid-driven hexanoic acid-producing strain and its yellow water fermentation regulation method

Abstract

This invention discloses a lactic acid-driven hexanoic acid-producing strain and its method for regulating the fermentation of yellow liquor, belonging to the field of microbial fermentation technology. The *Lactobacillus lactis* SCP250301 strain provided by this invention was isolated from the cellar mud of a strong-aroma baijiu distillery. Addressing the production pain point of unstable hexanoic acid yield due to significant fluctuations in the lactic acid to acetic acid ratio in yellow liquor with cellar age and season, this invention clarifies the optimal substrate ratio range for this strain: when the lactic acid:acetic acid carbon molar ratio is ≥5:1, the hexanoic acid yield remains at a high level; when the lactic acid:acetic acid carbon molar ratio is lower than 1:5, the hexanoic acid yield approaches 0, providing direct quantitative basis for substrate ratio regulation in the practical utilization of yellow liquor. This invention also discloses a method for enhancing hexanoic acid production in the baijiu yellow liquor system using the above strain and corresponding microbial agents, which can be applied to the resource utilization of high-lactic acid yellow liquor, enhanced cultivation of artificial cellar mud, and increasing hexanoic acid and reducing lactic acid in strong-aroma baijiu brewing, showing good industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a lactic acid-driven hexanoic acid-producing strain and its yellow water fermentation regulation method. Background Technology

[0002] Hexanoic acid and its esterification product, ethyl hexanoate, are important flavor compounds in strong-aroma baijiu, contributing to its main aroma and harmonious taste. They are defined as characteristic flavor components in GB / T10781.1-2021, "Quality Requirements for Baijiu Part 1: Strong-Aroma Baijiu." The formation and accumulation level of hexanoic acid in fermentation pit mud directly affects the stylistic typicality and product quality stability of strong-aroma baijiu. Therefore, achieving stable and efficient biosynthesis of hexanoic acid is a crucial technical means to improve the quality of strong-aroma baijiu.

[0003] In existing production processes, hexanoic acid is mainly generated through the metabolic activity of hexanoic acid-producing functional bacteria in the pit mud. These microorganisms can utilize substrates such as glucose, lactic acid, and ethanol to gradually generate butyric acid and hexanoic acid through a carbon chain elongation pathway. However, due to the complexity of the pit mud system's microecology and fluctuations in substrate composition, the hexanoic acid generation process suffers from unstable yields and significant efficiency fluctuations, making precise control difficult.

[0004] Yellow water is a liquid byproduct produced during the solid-state fermentation of strong-aroma baijiu, characterized by its stable source and large yield. Typically, approximately 0.30 to 0.40 tons of yellow water are produced for every ton of baijiu produced. Yellow water is rich in organic acids such as lactic acid, acetic acid, butyric acid, and a small amount of hexanoic acid, as well as alcohols, esters, and nitrogen-containing compounds, possessing the potential for reuse as a fermentation substrate. Lactic acid and acetic acid are the main organic acid components, and their content and proportion fluctuate significantly depending on the age of the fermentation pit and production conditions.

[0005] At present, the treatment of yellow water is still mainly based on standard discharge or low-value utilization, which fails to give full play to its utilization value as an organic acid resource. In recent years, the use of yellow water to promote the biosynthesis of hexanoic acid has gradually attracted attention, but the following technical problems still exist in practical applications: (1) Existing studies are mostly based on mixed substrate systems, and there is a lack of systematic characterization of the metabolic behavior of single strains under specific substrate conditions, resulting in unclear performance evaluation of strains, which is not conducive to process scale-up and stable application; (2) There is a lack of systematic quantitative research on the metabolic response mechanism of hexanoic acid producing bacteria under different lactic acid and acetic acid ratios, which makes it difficult to guide the optimization and control of key substrate ratios in yellow water, thereby affecting the hexanoic acid production efficiency and process stability; (3) There is a lack of fermentation process systems that match specific functional strains, resulting in low conversion efficiency and large product fluctuations in the process of yellow water resource utilization.

[0006] Therefore, there is an urgent need to develop a hexanoic acid-producing bacterium capable of efficiently utilizing key substrates such as lactic acid in yellow water, and to establish a fermentation process adapted to its metabolic characteristics, so as to achieve stable and efficient production of hexanoic acid and high-value utilization of yellow water. Based on this, this invention screened a hexanoic acid-producing bacterium with highly efficient lactic acid metabolism from baijiu cellar mud, systematically characterized its metabolic characteristics under different substrate conditions, and further constructed a corresponding fermentation process system, thereby providing a new technical solution for the targeted and enhanced production of hexanoic acid in strong-aroma baijiu and the resource utilization of yellow water. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a hexanoic acid-producing strain and its application in the resource utilization of yellow water, thereby solving the technical problems of unstable hexanoic acid production due to fluctuations in the lactic acid / acetic acid ratio in yellow water and the lack of a basis for substrate ratio regulation. This invention obtains a hexanoic acid-producing strain tolerant to low pH and capable of utilizing multiple carbon sources by screening from the fermentation mud of strong-aroma baijiu (Chinese liquor) cellars. It clarifies the acid production patterns and optimal substrate ratio range under different lactic acid / acetic acid ratios, thus providing technical support for the stable production of hexanoic acid in yellow water fermentation systems.

[0008] The *Lactobacillus lactis* strain SCP250301 provided by this invention was isolated from the cellar mud of a strong-aroma baijiu distillery in Sichuan. It can grow stably in a weakly acidic environment with a pH of 5.0–6.5, which is compatible with the actual pH range of yellow water, allowing for application without significant pH adjustments. It can utilize various sugars such as glucose, sucrose, fructose, lactose, D-mannitol, and soluble starch as carbon sources for growth. This strain can directly and efficiently synthesize hexanoic acid using lactic acid as the main substrate, achieving a final hexanoic acid yield of approximately 9.08 g / L under high lactic acid concentration conditions.

[0009] To achieve this objective, this invention isolated and screened a strain of hexanoic acid-producing bacteria with excellent characteristics; it was identified and named *Lactobacillus lactis*. Caproicibacteriumlactatifermentans It is deposited at the Guangdong Institute of Microbiology, with accession number GDMCC NO: 68030.

[0010] Furthermore, the strain has the following technical characteristics: (1) It is tolerant to low pH. The optimal pH for this strain is between 5.0 and 6.5, which is compatible with the pH value of yellow water (usually between 4.0 and 5.5). Fermentation can be carried out without significant pH adjustment; (2) It has a strong ability to produce hexanoic acid. Under optimal fermentation conditions, the hexanoic acid yield can reach 9.08 g / L, which has significant potential for industrial application; (3) It has a broad substrate spectrum and can efficiently utilize a variety of carbon sources such as maltose, D-mannitol, glucose, fructose, sucrose, lactose, pyruvic acid, and soluble starch.

[0011] Furthermore, this invention investigated the fermentation performance of the strain under different carbon source conditions. Fermentation cultures were conducted using glucose, lactic acid, ethanol, and acetic acid as the sole carbon sources, respectively. Fermentation media were prepared with equimolar carbon amounts, and the carbon source concentration was 9–12 g / L. Anaerobic fermentation was carried out at 37°C for 12 days, and the hexanoic acid yield at the end of fermentation was measured. The results showed that the hexanoic acid yields in the four groups were 2.19 g / L, 1.63 g / L, 0.15 g / L, and 0.12 g / L, respectively. The hexanoic acid yield was significantly higher in the groups using glucose and lactic acid as carbon sources than in the acetic acid and ethanol groups, indicating that the strain can directly utilize the dominant substrate in yellow water to efficiently produce hexanoic acid.

[0012] Furthermore, to simulate the dynamic fluctuations of lactic acid and acetic acid in yellow water, carbon source systems with different lactic acid / acetic acid ratios were established. Each 1 L of the basal fermentation medium contained 5 g yeast extract, 5 g peptone, 1 g sodium dihydrogen phosphate, 0.5 g dipotassium hydrogen phosphate, 2 g ammonium sulfate, 0.2 g magnesium sulfate heptahydrate, 0.5 g cysteine ​​hydrochloride, 6 g / L glucose, and 3% (v / v) ethanol, sterilized by organic membrane filtration. 350 µL of the metal element mother liquor was autoclaved at 115℃ for 30 min. In one embodiment of the invention, the total carbon content of lactic acid and acetic acid was fixed at 1.5 mol, and seven treatment groups were set up: lactic acid:acetic acid = 1:0, 10:1, 5:1, 1:1, 1:5, 1:10, and 0:1. Activated bacterial solutions were inoculated at a 10% v / v inoculum, and anaerobic fermentation was carried out at 37℃ for 15 days. Hexanoic acid yield and flavor composition were then determined.

[0013] Furthermore, the lactic acid:acetic acid ratio significantly affected hexanoic acid yield. The highest hexanoic acid yield was achieved in the lactic acid:acetic acid = 1:0 group, reaching 9.08 g / L. The yields in the 10:1 and 5:1 groups were similar, exceeding 2.4 g / L. The hexanoic acid yield in the 1:1 group was significantly lower than the high lactic acid group, reaching approximately 1.71 g / L at the end. When the acetic acid ratio was further increased to lactic acid:acetic acid = 1:5, 1:10, and 0:1, the hexanoic acid yield approached 0 g / L. Gas chromatography-mass spectrometry analysis of the fermentation products from the group with the highest hexanoic acid yield detected 38 volatile flavor compounds, mainly including 11 esters, 9 alcohols, 7 ketones, 6 pyrazines, and 5 aldehydes.

[0014] The present invention also provides a microbial preparation containing the above-mentioned Lactobacillus SCP250301.

[0015] Preferably, the microbial preparation is a liquid or solid bacterial agent containing live cells of *Lactobacillus schreiberensis* SCP250301, or a fermentation broth containing *Lactobacillus schreiberensis* SCP250301; preferably, the live cells of *Lactobacillus schreiberensis* SCP250301 are freeze-dried *Lactobacillus schreiberensis* SCP250301 cells or immobilized *Lactobacillus schreiberensis* SCP250301 cells.

[0016] Preferably, the number of viable Lactobacillus lactis per milliliter or per gram of microbial agent reaches 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL and above.

[0017] The present invention also provides a method for simultaneously producing hexanoic acid and butyric acid, wherein the method comprises fermenting the above-mentioned *Lactobacillus lactis* SCP250301, or using the above-mentioned microbial agent, with glucose, lactic acid, acetic acid or ethanol as carbon source, to prepare hexanoic acid and butyric acid.

[0018] The present invention also provides a method for preparing hexanoic acid using yellow water. The method involves fermenting the above-mentioned *Lactobacillus lactis* SCP250301 or the above-mentioned microbial agent with yellow water as a substrate to obtain hexanoic acid.

[0019] The present invention also provides a fermentation method for producing hexanoic acid based on substrate ratio control. The method involves determining the initial concentrations of lactic acid and acetic acid in the system before actual fermentation, adjusting the carbon molar ratio of lactic acid to acetic acid to not less than 5:1, inoculating with the above-mentioned *Lactobacillus lactis* SCP250301, or using the above-mentioned microbial agent, and then preparing hexanoic acid through fermentation.

[0020] Preferably, the fermentation conditions are a temperature of 37°C, an inoculum size of 10% (v / v), and anaerobic static culture for 15 days.

[0021] The strains and control methods described in this invention can be applied to the fortification of hexanoic acid, the targeted production of hexanoic acid from yellow water resources, and the precise control of the flavor of strong-aroma baijiu in the production of strong-aroma baijiu.

[0022] Beneficial effects (1) Outstanding salt and acid tolerance. The strain's tolerance to NaCl reaches an upper limit of 25 g / L, significantly higher than the tolerance level of existing similar hexanoic acid-producing strains. The high inorganic salt content in yellow water allows the strain to maintain normal growth and acid production metabolism in the complex ionic environment of yellow water, eliminating the need for desalination pretreatment and reducing process costs. Furthermore, its optimal growth pH is 5.0–6.5, highly compatible with the typical pH range of yellow water, allowing direct inoculation and fermentation in the acidic environment of yellow water without significant pH adjustment, simplifying the pre-fermentation treatment process.

[0023] (2) The hexanoic acid producing strain provided by the present invention has typical lactic acid-preferred metabolic characteristics. In the simulated yellow water substrate fermentation system, the hexanoic acid production can reach 9.08 g / L, which is higher than the existing reported similar hexanoic acid producing strains. It has a better hexanoic acid synthesis advantage and provides a more efficient strain selection for hexanoic acid enhancement fermentation in yellow water resource utilization.

[0024] (3) The hexanoic acid producing strain provided by the present invention can not only efficiently generate hexanoic acid in the complex substrate fermentation system, but also synergistically promote the formation of a variety of volatile flavor substances such as alcohols, pyrazines and esters, thereby effectively improving the flavor complexity and layering of the fermentation products, and providing a reliable strain basis for the precise control of the flavor of strong-aroma baijiu.

[0025] Preservation of biological materials A strain of Lactobacillus lactis Caproicibacterium lactatifermentans SCP250301, taxonomic name is Caproicibacterium lactatifermentans It was deposited on April 2, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 68030, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology. Attached Figure Description

[0026] Figure 1 For the present invention, Lactobacillus lactis ( Caproicibacterium lactatifermentans ) Colony morphology diagram of SCP250301.

[0027] Figure 2 For the present invention, Lactobacillus lactis ( Caproicibacterium lactatifermentans Ethanol tolerance diagram of SCP250301.

[0028] Figure 3 For the present invention, Lactobacillus lactis ( Caproicibacterium lactatifermentans Salt tolerance diagram of SCP250301.

[0029] Figure 4 For the present invention, Lactobacillus lactis ( Caproicibacterium lactatifermentans Lactic acid tolerance diagram of SCP250301.

[0030] Figure 5 For the present invention, Lactobacillus lactis ( Caproicibacterium lactatifermentans Hexanoic acid tolerance diagram of SCP250301. Detailed Implementation

[0031] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, the specific embodiments, structures, features, and effects of the present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0032] The culture media involved in the following examples are as follows: 1L of CGMP medium contains: 10 g peptone, 10 g yeast extract, 10 g glucose, 5 g sodium acetate, 2 g ammonium sulfate, 1 g sodium dihydrogen phosphate, 0.5 g dipotassium hydrogen phosphate, 0.1 g magnesium sulfate heptahydrate, and 350 µL of metal element stock solution. It is used after autoclaving at 115℃ for 30 min.

[0033] 1L of basal culture medium contains: 5 g yeast extract, 5 g peptone, 1 g sodium dihydrogen phosphate, 0.5 g dipotassium hydrogen phosphate, 2 g ammonium sulfate, 0.2 g magnesium sulfate heptahydrate, 0.5 g cysteine ​​hydrochloride, 350 µL of metal element stock solution, 6 g glucose, 3% ethanol (v / v), and is autoclaved at 115℃ for 30 min.

[0034] Mother liquor of metal elements (1L): 15 g ferrous sulfate, 10 g zinc sulfate, 10 g calcium chloride, 10 g cobalt chloride.

[0035] Different carbon source media: Add the following single carbon sources to the basal medium: glucose, lactic acid, acetic acid, or ethanol. Use 12 g / L glucose as the carbon basis, and replace other carbon sources with an equal amount based on their carbon molar amounts. Autoclave at 115℃ for 30 min before use.

[0036] Simulated yellow water culture medium: Lactic acid and acetic acid were added to the basal culture medium. The total carbon content of lactic acid and acetic acid was fixed at 1.5 mol. Seven treatment groups were set up, including lactic acid:acetic acid = 1:0, 10:1, 5:1, 1:1, 1:5, 1:10, and 0:1.

[0037] The detection methods involved in the following embodiments are as follows: Glucose detection methods: Accurately weigh 0.01 g of anhydrous glucose dried at 105℃ to prepare a 1.00 mg / mL standard solution. Dilute this solution to prepare standard solutions of 100 µg / mL, 200 µg / mL, 300 µg / mL, 400 µg / mL, 500 µg / mL, 600 µg / mL, 700 µg / mL, 800 µg / mL, 900 µg / mL, and 1000 µg / mL. Add 1.5 mL of DNS reagent, incubate in a boiling water bath for 15 min, cool, and then dilute to 25 mL with distilled water. Measure the absorbance at 540 nm. Establish a standard curve with different glucose solution concentrations on the x-axis and absorbance values ​​on the y-axis.

[0038] Take 200 µL of sample solution, add 300 µL of DNS reagent, and mix well. Incubate in a boiling water bath for 5 min, then rapidly cool to room temperature (a cold water bath can be used). Add 1.5 mL of distilled water to dilute the solution and improve measurement sensitivity. Measure the absorbance (OD) at 540 nm. 540 Plot a standard curve and calculate the reducing sugar content in the sample.

[0039] The detection methods for short / medium chain fatty acids and ethanol are as follows: High-performance liquid chromatography (HPLC) was used to analyze short / medium-chain fatty acids in the fermentation system. The column was a Carbomix H. 0.5 mL of fermentation broth (which could be appropriately diluted) was centrifuged at 12000 rpm for 2 min to remove cells. The supernatant was filtered through a 0.22 µm filter membrane before analysis for short / medium-chain fatty acids (S / MCFAs). The mobile phase was 2.5 mmol / L dilute sulfuric acid solution, and the flow rate was 0.6 mL / min. 1 Column temperature 55℃.

[0040] Methods for detecting volatile flavor compounds: The determination was performed using a Shimadzu QP-2010SE gas chromatography-mass spectrometry (GC-MS) system. Sample preparation: After centrifuging the fermentation broth at 8000 rpm for 10 min, 3 mL of the supernatant was transferred to a 20 mL amber GC flask. 1 g of sodium chloride and 10 µL of 16 mg / L chromatographic grade R2-octanol internal standard solution were added. GC conditions: Injector temperature was 250℃; column specifications were VF-WAMS (60 m × 250 mm, 0.25 µm, Thermo, USA); helium was used as the carrier gas; splitless injection was used. The temperature program was as follows: hold at 40℃ for 2 min, increase to 130℃ at a rate of 3℃ / min, increase to 200℃ at a rate of 6℃ / min, and then increase to 230℃ at a rate of 8℃ / min and hold for 8 min. MS conditions: EI ionization source, ion source temperature 250℃, interface temperature 250℃, ion source energy 70 eV, electron source scan range 25-350 amu.

[0041] Example 1: Isolation and screening of Lactobacillus acidophilus The specific steps are as follows: 1. Isolation and screening of Lactobacillus acidophilus Using pit mud from a strong-aroma baijiu distillery in Sichuan as the experimental material, samples were rapidly transferred under low-temperature anaerobic conditions after sampling. CGMP culture medium was dispensed into 150 mL capped glass bottles, each containing 80 mL of liquid. The bottles were autoclaved (115℃, 30 min) and then placed in an anaerobic chamber for thorough deoxygenation. 10 g of pit mud sample was weighed and directly inoculated into a glass bottle containing the culture medium. The bottle was gently shaken to ensure thorough dispersion, and incubated at 37℃ for 5 days for subsequent plating and separation.

[0042] Add 1 mL of pit mud enrichment to 9 mL of sterile physiological saline, mix thoroughly, and then take 100 μL for a tenfold serial dilution until 10... -7 Take the dilution of 10... -3 10 -4 10 -5 10 -6 and 10 -7 200 μL of each bacterial suspension was inoculated onto pre-prepared solid culture plates and evenly spread using a sterile spreader. The plates were then incubated in an anaerobic incubator at 37°C for 5–14 days. Colony growth was observed regularly during this period, and representative single colonies were selected based on colony morphology for streak inoculation to obtain pure cultures. The purified strains were further inoculated into liquid culture medium and incubated at 37°C for 3–5 days to obtain stably growing single strains.

[0043] Using Sanger sequencing, a strain producing hexanoic acid was finally screened and named SCP250301.

[0044] 2. Molecular identification of Lactobacillus acidophilus Two mL of bacterial culture from the strain screened in Example 1 was collected by centrifugation at 8000 rpm for 5 min. Genomic DNA was extracted using a bacterial genomic DNA extraction kit. Using the extracted DNA as a template, PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-TACGGYTACCTTGTTACGACTT-3', SEQ ID NO.2) for the bacterial 16S rRNA gene. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, for a total of 35 cycles; final extension at 72℃ for 10 min. After verification by 1% agarose gel electrophoresis, the PCR products were sent to Chengdu Sangon Biotech Co., Ltd. for Sanger sequencing.

[0045] The obtained 16S rRNA gene sequence was submitted to the NCBI database for BLAST comparison. The results showed that the strain had a similarity of 99.85% with *Lactobacillus lactis*. Based on the colony morphology and physiological and biochemical characteristics, the strain was identified as *Lactobacillus lactis* and named *Lactobacillus lactis*. Caproicibacterium lactatifermentans SCP250301.

[0046] The 16S rDNA sequence is as follows (SEQ ID NO.3): Example 2: Performance determination of Lactobacillus SCP250301 1. Acid resistance test (1) Preparation of culture media with different pH values Using CGMP medium as a base, initial pH gradients of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 were established. These pH values ​​were adjusted to the target values ​​using 5 mol / L HCl or NaOH solution, and the pH deviation was controlled within ±0.1 after sterilization. The different pH media were aliquoted into 50 mL centrifuge tubes, with each tube containing 30 mL. Three biological replicates were performed for each pH treatment.

[0047] (2) Preparation of seed liquid The *Lactobacillus lactis* SCP250301 strain, stored at -80℃ in 25% glycerol cryovials, was inoculated into 5 mL of CGMP medium and statically activated at 37℃ for 24 h under anaerobic conditions, completing the first step of revival. Subsequently, a 10% (v / v) inoculum was transferred to 15 mL of CGMP medium for the first subculture, and anaerobically incubated at 37℃ for 24 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase (i.e., static incubation at 37℃ for 24 h), a second subculture was performed at a 10% (v / v) inoculum to 150 mL of CGMP medium, and anaerobically incubated until the logarithmic growth phase (i.e., static incubation at 37℃ for 24 h). The resulting bacterial culture was used as the seed culture for subsequent fermentation experiments.

[0048] (3) The activated seed culture was inoculated into the culture medium (different pH) obtained in step (1) at an inoculation rate of 10% (v / v), and anaerobic cultured at 37℃ for 5 days. Samples were taken at 0, 24, 48, 72, 96 and 120 h of fermentation, and frozen at -20℃ for subsequent OD measurement. 600 To characterize the growth of the strain.

[0049] OD of Lactobacillus lactis under different initial pH conditions 600 The changes are shown in Table 1.

[0050] Table 1: OD under different pH conditions 600

[0051] The results showed that the growth range of Lactobacillus strain was 4.5-9.0, indicating that this strain has strong acid and alkali tolerance.

[0052] It is worth noting that the pH of yellow water is usually between 4.0 and 5.5. The 5.0-5.5 range overlaps with the optimal pH range for the growth of the strain, indicating that the strain can maintain good growth within this pH range. For the pH range below 5.0, although it slightly exceeds the labeled growth range of the strain, considering the dynamic change characteristics of the pH of yellow water and the tolerance of the strain to boundary pH conditions, there is no need to make significant pre-adjustments to the pH of yellow water in practical applications.

[0053] Therefore, this strain has good tolerance and adaptability to the typical acidic environment of yellow water, and has the potential to be directly applied to actual production, thus having significant application value.

[0054] 2. Carbon source utilization spectrum (1) Preparation of culture medium In CGMP medium, glucose was replaced with 11 different carbon sources: arabinose, maltose, D-mannitol, fructose, sucrose, lactose, sodium pyruvate, ethanol, L-fucose, xylose, and soluble starch. The other components of the medium remained unchanged. Meanwhile, the original basal medium was used as the 12th carbon source medium to detect the carbon source utilization of the strains.

[0055] The above-mentioned different carbon source culture media (12 kinds) were dispensed into 50 mL centrifuge tubes, with each tube containing 30 mL. Three biological replicates were set up for each carbon source condition.

[0056] (2) Preparation of seed liquid The *Lactobacillus lactis* SCP250301 strain, stored at -80℃ in 25% glycerol cryovials, was inoculated into 5 mL of CGMP medium and statically activated at 37℃ for 24 h under anaerobic conditions, completing the first step of revival. Subsequently, a 10% (v / v) inoculum was transferred to 15 mL of CGMP medium for the first subculture, and anaerobically incubated at 37℃ for 24 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase (i.e., static incubation at 37℃ for 24 h), a second subculture was performed at a 10% (v / v) inoculum to 150 mL of CGMP medium, and anaerobically incubated until the logarithmic growth phase (i.e., static incubation at 37℃ for 24 h). The resulting bacterial culture was used as the seed culture for subsequent fermentation experiments.

[0057] (3) The activated seed culture was inoculated into the culture medium with different carbon sources obtained in step (1) at an inoculation rate of 10% (v / v), and anaerobic cultured at 37℃ for 5 days. Samples were taken at 0, 24, 48, 72, 96 and 120 h of fermentation, and frozen at -20℃. OD was subsequently measured. 600 To characterize the growth of the strain.

[0058] The growth of *Lactobacillus lactis* under various carbon source conditions is shown in Table 2.

[0059] Table 2: OD under different carbon source conditions 600

[0060] The results showed that the strain grew relatively weakly in a culture medium using arabinose, ethanol, and L-fucose as carbon sources, with low OD. 600 The peak values ​​were 0.52, 0.3 and 0.8, respectively, indicating that the strain had limited ability to utilize the aforementioned carbon sources.

[0061] Under the conditions of the remaining 9 carbon sources, OD 600 All values ​​were greater than 1.5, and all strains were able to grow normally and reach high biomass, indicating that the strain has a broad carbon source utilization spectrum. Among them, the strain grew most vigorously when maltose and lactose were used as carbon sources. 600 The values ​​reached as high as 2.6 and 2.3, respectively, indicating that the aforementioned carbon sources are the dominant substrates utilized by this strain.

[0062] Yellow water is rich in various organic acids and sugars such as lactic acid, acetic acid, and glucose. The strain has good utilization ability of the above substrates, indicating that the strain has good adaptability to the complex substrate composition of yellow water, can grow efficiently in the yellow water fermentation system and play the role of producing hexanoic acid, and has the potential to be directly applied to the resource-based fermentation of yellow water.

[0063] 3. Ethanol tolerance (1) Preparation of culture medium Anhydrous ethanol (sterilized by organic membrane filtration) was added to CGMP medium as the basal medium. The ethanol concentrations of the different media were 0%, 2%, 4%, 6%, 8%, and 10% (v / v).

[0064] (2) Prepare seed solution according to the method described in step 1; The prepared Lactobacillus SCP250301 seed culture was inoculated into culture media with different ethanol concentrations obtained in step (1) at an inoculation rate of 10% (v / v), and anaerobic static culture was carried out at 37°C for 3 days. The OD value of the fermentation broth on the last day was measured at a wavelength of 600 nm.

[0065] The results showed that, Figure 2 As shown, the OD of the strain was between 0% and 4% (v / v) of ethanol. 600 No significant changes indicate that the strain is growing well at this stage; when the ethanol concentration reaches 6-10% (v / v), the OD... 600 A sharp decline; Even under conditions where the ethanol concentration is as high as 10% (v / v), the strain still maintains a certain growth capacity, indicating that the upper limit of ethanol tolerance of the strain of the present invention can reach 10% (v / v), and it has strong ethanol tolerance.

[0066] 4. Salt tolerance (1) Preparation of culture medium Sodium chloride was added to CGMP medium as the basal medium, and the sodium chloride concentrations of the different media were 2.5, 5, 10, 15, 20, and 25 (g / L).

[0067] (2) Prepare seed solution according to the method described in step 1; The prepared Lactobacillus SCP250301 seed culture was inoculated into culture media with different sodium chloride concentrations obtained in step (1) at an inoculation rate of 10% (v / v), and anaerobic static culture was carried out at 37°C for 3 days. The OD value of the fermentation broth on the last day was measured at a wavelength of 600 nm.

[0068] The results showed that, Figure 3 As shown, with increasing salt concentration, OD 600 The value showed a gradually decreasing trend. When the salt concentration increased from 2.5 g / L to 25 g / L, the OD value decreased. 600 It dropped from 2.25 to around 0.76.

[0069] It is worth noting that the salt tolerance limit of similar hexanoic acid-producing strains in existing reports is usually no more than 10 g / L, while the salt tolerance limit of the strain of this invention is as high as 25 g / L, which is significantly better than the reported similar strains, indicating that the strain of this invention has outstanding salt tolerance.

[0070] 5. Lactic acid tolerance (1) Preparation of culture medium Lactic acid was added to CGMP medium as the basal medium, and the lactic acid concentrations in the different media were 2, 5, 10, 20, 40, 60 and 80 (g / L).

[0071] (2) Prepare seed solution according to the method described in step 1; The prepared Lactobacillus SCP250301 seed culture was inoculated into culture media with different lactic acid concentrations obtained in step (1) at an inoculation rate of 10% (v / v), and anaerobic static culture was carried out at 37°C for 3 days. The OD value of the fermentation broth on the last day was measured at a wavelength of 600 nm.

[0072] The results show that, according to Figure 4 Within the acid concentration range of 2 g / L to 10 g / L, the OD of the strain... 600The value did not change significantly, but when the acid concentration increased to 20 g / L, the OD value increased. 600 The value began to show a downward trend; Even under extreme conditions with acid concentrations as high as 60 g / L to 80 g / L, the strain's OD 600 The value can still be maintained between 0.1 and 0.3, indicating that the strain has a strong tolerance to lactic acid.

[0073] 6. Tolerance to hexanoic acid (1) Preparation of culture medium Hexanoic acid was added to CGMP medium as the basal medium, and the concentrations of hexanoic acid in the different media were 0, 2, 4, 6, 8, 10, and 12 (g / L).

[0074] (2) Prepare seed solution according to the method described in step 1; The prepared Lactobacillus SCP250301 seed culture was inoculated into culture media with different hexanoic acid concentrations obtained in step (1) at an inoculation rate of 10% (v / v), and anaerobic static culture was carried out at 37°C for 3 days. The OD value of the fermentation broth on the last day was measured at a wavelength of 600 nm.

[0075] The results show that, according to Figure 5 Within a hexanoic acid concentration range of 0 g / L to 12 g / L, this strain maintained normal growth. Under low acid concentrations (≤4 g / L), the strain's growth activity was largely unaffected, with the OD value remaining above 1.0. As the acid concentration further increased, the strain's growth activity showed a slow downward trend, but even under extreme conditions with an acid concentration as high as 8 g / L, the OD value could still be maintained above 0.4, indicating that this strain has strong tolerance to this acid and possesses good potential for industrial applications.

[0076] Example 3: Determination of hexanoic acid fermentation performance of Lactobacillus hexanolyte SCP250301 under different carbon source conditions The specific steps are as follows: (1) Preparation of culture medium The following carbon sources were added to the basal medium (CGMP medium without glucose): glucose, lactic acid, acetic acid, and ethanol (four in total). Using 12 g / L glucose as the carbon baseline, the other carbon sources were replaced by equal amounts of carbon molarity, resulting in lactic acid at 12 g / L, acetic acid at 12 g / L, and ethanol at 9 g / L.

[0077] The culture media with different carbon sources were dispensed into 150 mL glass bottles, with each bottle containing 80 mL. Three biological replicates were performed for each carbon source condition. The culture media were sterilized at 115°C for 30 min, cooled to room temperature, and then purged with nitrogen to remove oxygen.

[0078] (2) Preparation of seed liquid The *Lactobacillus lactis* SCP250301 strain, stored at -80℃ in 25% glycerol cryovials, was inoculated into 5 mL of CGMP medium and statically activated at 37℃ for 24 h under anaerobic conditions, completing the first step of revival. Subsequently, a 10% (v / v) inoculum was transferred to 15 mL of CGMP medium for the first subculture, and anaerobically incubated at 37℃ for 24 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase (i.e., static incubation at 37℃ for 24 h), a second subculture was performed at a 10% (v / v) inoculum to 150 mL of CGMP medium, and anaerobically incubated until the logarithmic growth phase (i.e., static incubation at 37℃ for 24 h). The resulting bacterial culture was used as the seed culture for subsequent fermentation experiments.

[0079] (3) The Lactobacillus SCP250301 seed culture obtained in step (2) was inoculated into the culture media with different carbon sources obtained in step (1) at an inoculation rate of 10% (v / v), and anaerobic static culture was carried out at 37℃ for 12 days, with samples taken periodically. After fermentation, the content of short-chain and medium-chain fatty acids was determined by high performance liquid chromatography (HPLC).

[0080] The results are shown in Table 3 below: Table 3: Substrate consumption, short- and medium-chain fatty acid accumulation, growth, and pH at fermentation endpoints (day 12) for different carbon sources

[0081] The results showed that when glucose was used as the carbon source, the yield of hexanoic acid could reach 2.19 g / L; When lactic acid is used as the carbon source, the yield of hexanoic acid is 1.63 g / L; When ethanol is used as the carbon source, the yield of hexanoic acid is 0.15 g / L; The yield of hexanoic acid was lowest when acetic acid was used as the carbon source, at 0.12 g / L.

[0082] The yield of hexanoic acid varied under different carbon source conditions, indicating that the type of carbon source has an important influence on the hexanoic acid synthesis and metabolism of the strain.

[0083] The above results demonstrate that the *Lactobacillus lactis* SCP250301 strain can directly and efficiently synthesize hexanoic acid from lactic acid, with hexanoic acid yield higher when lactic acid is used as the carbon source than when ethanol or acetic acid is used. Given that lactic acid is the main organic acid component in the yellow water of strong-aroma baijiu, with a concentration typically of 30–80 g / L, far exceeding that of acetic acid (2–10 g / L), this strain can directly use lactic acid from the yellow water as a substrate for hexanoic acid fermentation without the need for additional exogenous carbon sources. This reduces fermentation costs, simplifies the process, and provides a technical basis for the direct application of hexanoic acid production from yellow water resources, demonstrating high practical application value in the production of strong-aroma baijiu.

[0084] Example 4: Effect of lactic acid / acetic acid substrate ratio on the fermentation performance of *Lactobacillus lactis* producing hexanoic acid The specific steps are as follows: (1) Preparation of fermentation media with different lactic acid / acetic acid ratios To simulate the effect of substrate ratio fluctuations in yellow water on the hexanoic acid production capacity of the strain, the glucose concentration was fixed at 6 g / L and the ethanol concentration at 3% (v / v) in the basal medium to reflect the actual average levels of glucose and ethanol in the yellow water.

[0085] 1L of basal culture medium contains: 5 g yeast extract, 5 g peptone, 1 g sodium dihydrogen phosphate, 0.5 g dipotassium hydrogen phosphate, 2 g ammonium sulfate, 0.2 g magnesium sulfate heptahydrate, 0.5 g cysteine ​​hydrochloride, 350 µL of metal element stock solution, 6 g glucose, and 3% ethanol (v / v); Metal element stock solution (1L): 15 g ferrous sulfate, 10 g zinc sulfate, 10 g calcium chloride, and 10 g cobalt chloride.

[0086] Based on this, lactic acid and acetic acid were added to the above culture medium. With the total carbon molar amount of lactic acid and acetic acid being kept constant (1.5 mol C / L) as a constraint, the single variable control was achieved by adjusting the relative ratio of lactic acid and acetic acid. A total of 7 treatment groups were set up. The carbon molar ratio (mol / mol) of lactic acid to acetic acid in each group was as follows: 1:0 (pure lactic acid group), 10:1, 5:1, 1:1, 1:5, 1:10, and 0:1 (pure acetic acid group). The corresponding lactic acid addition amounts were 45 g / L, 40.9 g / L, 37.5 g / L, 22.5 g / L, 7.5 g / L, 4.1 g / L, and 0 g / L, respectively, and the acetic acid addition amounts were 0 g / L, 4.1 g / L, 7.5 g / L, 22.5 g / L, 37.5 g / L, 40.9 g / L, and 45 g / L, respectively. The above treatment group design covers the full ratio gradient from pure lactic acid to pure acetic acid, which can systematically reflect the influence of the dynamic fluctuation of the lactic acid / acetic acid ratio in yellow water on the synthesis of hexanoic acid.

[0087] The different culture media with the above ratios were dispensed into 150 mL glass bottles, with each bottle containing 80 mL. Each treatment group had 3 biological replicates. The culture media of each group were first subjected to nitrogen purging to maintain a strictly anaerobic environment, and then sterilized at 115°C for 30 min.

[0088] (2) Preparation of seed liquid The *Lactobacillus lactis* SCP250301 strain, stored at -80℃ in 25% glycerol cryovials, was inoculated into 5 mL of CGMP medium and statically activated at 37℃ for 24 h under anaerobic conditions, completing the first step of revival. Subsequently, a 10% (v / v) inoculum was transferred to 15 mL of CGMP medium for the first subculture, and anaerobically incubated at 37℃ for 24 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase (i.e., static incubation at 37℃ for 24 h), a second subculture was performed at a 10% (v / v) inoculum to 150 mL of CGMP medium, and anaerobically incubated until the logarithmic growth phase (i.e., static incubation at 37℃ for 24 h). The resulting bacterial culture was used as the seed culture for subsequent fermentation experiments.

[0089] (3) The *Lactobacillus SCP250301* seed culture obtained in step (2) was inoculated into fermentation media with different lactic acid / acetic acid ratios at an inoculation rate of 10% (v / v), and anaerobic cultured at 37°C for 15 days, with samples taken periodically. After fermentation, the content of short / medium chain fatty acids was detected by high performance liquid chromatography (HPLC), and the composition of volatile flavor compounds was detected by gas chromatography-mass spectrometry (GC-MS).

[0090] The substrate consumption and acid production results for each treatment group are shown in Table 4.

[0091] Table 4: Fermentation endpoints for different groups: consumption of lactic acid and acetic acid, accumulation of short- and medium-chain fatty acids, growth, and pH.

[0092] The results showed that in the pure lactic acid group (lactic acid:acetic acid = 1:0), lactic acid consumption reached 35.18 g / L, and the hexanoic acid yield at the end of fermentation was approximately 9.08 g / L, the highest among the seven groups, while the peak butyric acid yield was approximately 2.91 g / L. In the high lactic acid group, the lactic acid:acetic acid = 10:1 and 5:1 groups had hexanoic acid yields of 2.66 g / L and 2.48 g / L at the end of fermentation, respectively, indicating that under lactic acid-dominant substrate conditions, the presence of a small amount of acetic acid did not significantly promote hexanoic acid synthesis. In the equal ratio group (lactic acid:acetic acid = 1:1), the hexanoic acid yield at the end of fermentation was approximately 1.71 g / L, showing a significant decrease in the higher lactic acid group, while butyric acid accumulated simultaneously to approximately 1.81 g / L. In the high acetic acid group and the pure acetic acid group (lactic acid:acetic acid = 1:5, 1:10 and 0:1), the utilization of acetic acid was extremely limited with further increases in the proportion of acetic acid. The yield of hexanoic acid during the entire fermentation process approached 0 g / L, and the maximum accumulation of butyric acid was only about 0.8 g / L. The metabolic activity of the strain was significantly inhibited, indicating that high concentrations of acetic acid have a significant inhibitory effect on the hexanoic acid production metabolism of the strain.

[0093] In summary, the hexanoic acid yield in all seven treatments decreased significantly with decreasing lactic acid ratio, indicating that the lactic acid / acetic acid ratio had a decisive impact on hexanoic acid synthesis: when the lactic acid:acetic acid ratio was ≥5:1, the hexanoic acid yield was ≥2.4 g / L; when the ratio decreased to 1:1, the yield dropped to 1.7 g / L; and when the ratio was ≤1:5, the yield approached 0 g / L. These results suggest that lactic acid is a key substrate for hexanoic acid production by *Lactobacillus lactis*, and an excessively high acetic acid ratio significantly inhibits hexanoic acid synthesis. Therefore, in practical yellow water fermentation, it is recommended to test the lactic acid to acetic acid ratio in the yellow water before fermentation starts and control the lactic acid / acetic acid ratio above 5:1. If necessary, adjustments can be made by adding lactic acid or diluting acetic acid to ensure stable and efficient hexanoic acid production.

[0094] (4) Gas chromatography-mass spectrometry (GC-MS) was used to analyze the fermentation endpoint products of the pure lactic acid group (lactic acid:acetic acid = 1:0) with the highest hexanoic acid yield.

[0095] A total of 38 volatile flavor compounds were detected, mainly including 11 esters, 9 alcohols, 7 ketones, 6 pyrazines, and 5 aldehydes. Typical compounds and their contents are shown in Table 5. (Compared with existing information on...) Caproicibacterium lactatifermentans Previous studies have mainly focused on the different formation of medium-chain fatty acids such as hexanoic acid. This invention further reveals that this strain can form a more complex system of volatile flavor compounds during fermentation.

[0096] Table 5: Content of volatile aroma compounds in pure lactic acid fermentation products from *Lactobacillus lactis*

[0097] The results show: Esters were the most abundant and prominent flavor components in this fermentation system. Isoamyl nitrite was the most abundant, with a concentration of 183.74 mg / L, exhibiting a distinct fruity aroma; followed by ethyl hexanoate, with a concentration of 9.18 mg / L, possessing a typical tropical fruit aroma and being an important characteristic flavor compound of strong-aroma baijiu. The large accumulation of these esters is closely related to the continuous production of hexanoic acid and other medium- and short-chain fatty acids during fermentation, indicating that the strain can promote the simultaneous synthesis of ester flavor compounds while producing hexanoic acid. Among alcohols, 1-hexadecaneol was the most abundant, with a typical rose aroma, at a concentration of 3.17 mg / L; followed by 2-nonyl alcohol, with a concentration of 1.40 mg / L, exhibiting rose and orange aromas.

[0098] It is noteworthy that various pyrazine compounds were detected in the fermentation system of the strain of this invention. These substances typically impart flavor characteristics such as nutty and roasted aromas, but they have rarely been reported or have not been reported in existing studies on this strain or related elongated strains. This indicates that the strain of this invention has a flavor metabolic pathway different from known strains, thereby enabling it to form fermentation products with complex aroma characteristics.

[0099] In summary, the strain described in this invention can not only efficiently synthesize hexanoic acid, but also simultaneously produce a variety of volatile flavor compounds that are not seen in existing technologies or whose content is significantly increased, forming a complex flavor system characterized by ester aroma, alcohol aroma, and nutty aroma. When applied to the resource-based fermentation process of yellow water, it can significantly improve the overall flavor quality of the fermentation system while enhancing hexanoic acid production, providing a new technical approach for the targeted regulation of the flavor of strong-aroma baijiu.

[0100] 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. A strain of *Lactobacillus lactis* ( Caproicibacterium lactatifermentans SCP250301, characterized in that, The Lactobacillus lactis is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 68030 and deposit date of April 2, 2026.

2. The *Lactobacillus lactis* SCP250301 according to claim 1, characterized in that, The growth pH range of *Lactobacillus lactis* SCP250301 is 4.5–9.0; *Lactobacillus lactis* SCP250301 utilizes one or more of the following as carbon sources for growth: maltose, D-mannitol, glucose, fructose, sucrose, lactose, pyruvate, soluble starch, and xylose; the ethanol concentration tolerated by *Lactobacillus lactis* SCP250301 is 0–10% (v / v); the salt concentration tolerated by *Lactobacillus lactis* SCP250301 is 2.5–25 g / L; the lactic acid concentration tolerated by *Lactobacillus lactis* SCP250301 is 2–80 g / L; and the hexanoic acid concentration tolerated by *Lactobacillus lactis* SCP250301 is 0–12 g / L.

3. A microbial preparation containing *Lactobacillus lactis* SCP250301 as described in claim 1 or 2.

4. The microbial preparation according to claim 3, characterized in that, The microbial preparation is a liquid or solid bacterial agent containing live cells of *Lactobacillus schreiberensis* SCP250301, or a fermentation broth containing *Lactobacillus schreiberensis* SCP250301; preferably, the live cells of *Lactobacillus schreiberensis* SCP250301 are freeze-dried *Lactobacillus schreiberensis* SCP250301 cells or immobilized *Lactobacillus schreiberensis* SCP250301 cells.

5. The microbial agent according to claim 3 or 4, characterized in that, The microbial agent contains 1×10⁶ live Lactobacillus. 8 CFU / g or 1×10 8 CFU / mL and above.

6. A method for simultaneously producing hexanoic acid and butyric acid, characterized in that, The method involves fermenting *Lactobacillus spp.* SCP250301 as described in claim 1 or 2, or using any of the microbial agents described in claims 3 to 5, with glucose, lactic acid, acetic acid, or ethanol as the carbon source, to prepare hexanoic acid and butyric acid.

7. A method for preparing hexanoic acid using yellow water, characterized in that, The method involves fermenting *Lactobacillus spp.* SCP250301 as described in claim 1 or 2, or using any of the microbial agents described in claims 3 to 5, with yellow water as the substrate, to obtain hexanoic acid.

8. A fermentation method for hexanoic acid production based on substrate ratio regulation, characterized in that, The method involves determining the initial concentrations of lactic acid and acetic acid in the system before actual fermentation, adjusting the carbon molar ratio of lactic acid to acetic acid to not less than 5:1, inoculating with *Lactobacillus lactis* SCP250301 as described in claim 1 or 2, or using any of the microbial agents described in claims 3 to 5, and then fermenting to obtain hexanoic acid.

9. The method according to claim 8, characterized in that, The fermentation conditions were: temperature 37°C, inoculum size 10% (v / v), and anaerobic static culture for 15 days.

10. The application of *Lactobacillus spp.* SCP250301 as described in claim 1 or 2, or the application of any of the microbial agents described in claims 3 to 5 in the strengthening of cellar mud for strong-aroma baijiu, the directional fermentation of yellow water to produce hexanoic acid, or the industrial production of hexanoic acid.

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