An acetic acid-tolerant hexanoic acid-producing strain and its application in enhanced fermentation of yellow water

Abstract

This invention discloses an acetic acid-tolerant hexanoic acid-producing strain and its application in enhanced fermentation of yellow water, belonging to the field of microbial fermentation technology. The *Clostridium krillion* GDMCC NO: 68031 strain of this invention can grow at pH 4.0–9.0 and can utilize carbon sources such as arabinose, fructose, lactose, ethanol, L-fucose, and glucose for growth and metabolism, exhibiting broad substrate utilization capabilities. The strain exhibits typical acetic acid-biased metabolic characteristics, with the highest hexanoic acid yield (up to 6.46 g / L) when acetic acid is the sole carbon source. In a composite fermentation system simulating yellow water substrate composition, the strain shows no significant difference in hexanoic acid yield across a wide range of lactic acid / acetic acid ratios, demonstrating high adaptability to substrate ratio fluctuations and simultaneously promoting the production of various volatile flavor compounds such as alcohols, pyrazines, and esters.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology and relates to an acetic acid-tolerant hexanoic acid-producing strain and its application in enhanced fermentation of yellow water. Background Technology

[0002] Strong-aroma baijiu is one of the most produced and widely consumed baijiu types in my country. Ethyl hexanoate is its core characteristic aroma component, and its content and proportion directly determine the typical style and quality grade of strong-aroma baijiu. Ethyl hexanoate is produced by the esterification reaction of hexanoic acid and ethanol. The efficient synthesis of hexanoic acid is a key step in the formation of the flavor and quality of strong-aroma baijiu, and the hexanoic acid-producing functional microorganisms in the cellar mud and fermentation system are the core driving force for hexanoic acid synthesis.

[0003] Yellow water is an important byproduct produced during the solid-state fermentation of strong-aroma baijiu, accumulating in large quantities at the bottom of the fermentation pits after each round of fermentation. Rich in lactic acid, acetic acid, glucose, and other organic acids and sugars, yellow water serves as a natural substrate for hexanoic acid-producing microorganisms, possessing high resource utilization value. Using yellow water as a raw material for hexanoic acid-enhanced fermentation not only facilitates the resource utilization of yellow water but also provides high-quality hexanoic acid-producing bacterial solution for baijiu production, thus having significant economic and production value.

[0004] However, the following problems still exist in the research and application of hexanoic acid fermentation for the resource utilization of yellow water: (1) The composition of yellow water substrate fluctuates greatly, and the existing hexanoic acid producing strains are not adaptable enough. The concentration and ratio of lactic acid and acetic acid in yellow water are affected by various factors such as the conditions of the fermentation pit, the fermentation cycle, and the seasonal temperature, and the batch-to-batch fluctuations are significant. Existing hexanoic acid producing strains are generally sensitive to the substrate ratio. Once the substrate ratio deviates from its suitable range, the hexanoic acid production capacity will decrease significantly, making it difficult to adapt to the dynamic changes in the composition of yellow water substrate. (2) The substrate pretreatment process is complicated, and the cost of industrial application is high. In order to maintain the stable acid production performance of hexanoic acid producing strains, it is often necessary to test each batch of yellow water and adjust the substrate ratio accordingly in actual production. The operation is cumbersome and costly, and it is difficult to continuously and stably implement in large-scale industrial production, which restricts the promotion and application of yellow water resource utilization fermentation. (3) Existing strains have poor tolerance to high acetic acid substrate environment, and the substrate utilization rate is low. Acetic acid is one of the organic acid components with high content in yellow water, and the acetic acid concentration in some batches of yellow water is relatively high. Existing hexanoic acid-producing strains exhibit significantly inhibited acid production under high acetic acid conditions, failing to effectively utilize the acetic acid resources in yellow water, resulting in low substrate utilization and limiting the actual production efficiency of hexanoic acid. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a hexanoic acid-producing strain that is highly adaptable to fluctuations in substrate ratios. This strain can maintain stable hexanoic acid production within a wide range of lactic acid / acetic acid ratios. It can still achieve effective hexanoic acid synthesis in complex substrate systems with high acetic acid content. It can be directly applied without significant pretreatment of the yellow water substrate ratio, providing a more robust and easier-to-operate strain for hexanoic acid enhancement fermentation in yellow water resources.

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a caproic acid-producing strain with an acetic acid-preferred metabolic characteristic, identified and named *Clostridium kuristegi* (…). Clostridium kluyverii The sample is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 68031 and deposit date of April 2, 2026.

[0007] In one embodiment of the present invention, the growth pH range of Clostridium krillion SCP250302 is 4.0 to 9.0; the Clostridium krillion SCP250302 utilizes one or more of arabinose, fructose, lactose, ethanol, L-fucose, and glucose as a carbon source for growth; the ethanol concentration tolerated by the Clostridium krillion SCP250302 is 0 to 10% (v / v); the salt concentration tolerated by the Clostridium krillion SCP250302 is 2.5 to 25 g / L; the acetic acid concentration tolerated by the Clostridium krillion SCP250302 is 2 to 80 g / L; and the hexanoic acid concentration tolerated by the Clostridium krillion SCP250302 is 0 to 12 g / L.

[0008] The present invention also provides a microbial inoculant containing the above-mentioned strains, wherein the viable count of the strains in each milliliter or per gram of the microbial inoculant is not less than 1 × 10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0009] The strain has the following excellent characteristics: (1) It is tolerant to low pH, with the optimal growth pH being 5.0~6.5, and can maintain growth activity in the pH range of 4.0~9.0; (2) It has a wide range of carbon source utilization, and can effectively utilize 6 of the 12 carbon source conditions for growth and metabolism; (3) It has typical acetic acid-preferred metabolic characteristics, and in the single substrate system with acetic acid as the only carbon source, the hexanoic acid yield is the highest, reaching 6.46 g / L, which is significantly higher than other carbon source conditions.

[0010] In one embodiment of the present invention, the microbial preparation is a liquid or solid bacterial agent containing live cells of Clostridium krill SCP250302, or a fermentation broth containing Clostridium krill SCP250302.

[0011] In one embodiment of the present invention, the Clostridium krillion SCP250302 live cells are freeze-dried Clostridium krillion SCP250302 mycelia or immobilized Clostridium krillion SCP250302 cells.

[0012] In one embodiment of the present invention, a full-scale gradient fermentation system with a lactic acid:acetic acid carbon molar ratio ranging from 1:0 to 0:1 was constructed in a composite fermentation system simulating a yellow water substrate composition, with a constant total carbon molar amount of lactic acid and acetic acid (1.5 mol C / L) as a constraint. The glucose concentration was fixed at 6 g / L and the ethanol concentration at 3% (v / v). The strain was inoculated at a 10% (v / v) inoculum and cultured anaerobically at 37°C for 15 days. The results showed that the strain achieved the highest hexanoic acid yield of 12.51 g / L under the lactic acid:acetic acid = 1:1 condition. In treatments with a higher acetic acid ratio (lactic acid:acetic acid ≤ 1:1), the hexanoic acid yield did not differ significantly, remaining above 9 g / L. This indicates that the strain has a high adaptability to fluctuations in the lactic acid / acetic acid substrate ratio and can achieve stable hexanoic acid synthesis over a wide substrate ratio range. The above characteristics are significantly different from those of strains that require the lactic acid / acetic acid ratio to be adjusted to a specific range for efficient acid production. These strains can be directly applied to fermentation without pre-adjusting the substrate ratio, simplifying the process and reducing reliance on pretreatment operations.

[0013] In one embodiment of the present invention, GC-MS analysis of the fermentation endpoint product of the lactic acid:acetic acid (1:1 ratio) group, which yielded the highest hexanoic acid, revealed 32 volatile flavor compounds, mainly including 13 alcohols, 3 aldehydes, 2 ketones, 8 pyrazines, and 6 esters. Hexanol and octanol were the main alcohols, 2,5-dimethylpyrazine was the main pyrazine, and ethyl hexanoate was the main ester. The simultaneous accumulation of these multiple flavor compounds indicates that the strain possesses strong carbon chain elongation and derivatization capabilities in the complex substrate fermentation system, enabling it to efficiently synthesize hexanoic acid while simultaneously promoting the generation of various alcohols and pyrazines, significantly enriching the flavor composition of the fermentation product.

[0014] The strains and fermentation methods described in this invention can be applied to resource-based hexanoic acid enhanced fermentation in yellow water systems with large fluctuations in substrate composition or high acetic acid content, targeted enhancement of hexanoic acid and flavor substances in the production of strong-aroma baijiu, and the development of simplified hexanoic acid fermentation processes that do not require substrate ratio pretreatment.

[0015] The present invention also provides a method for simultaneously producing hexanoic acid and butyric acid, wherein the method comprises fermenting the above-mentioned Clostridium krill SCP250302, 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.

[0016] The present invention also provides a method for preparing hexanoic acid using yellow water. The method involves fermenting the Clostridium krill SCP250302 or the above-mentioned microbial agent with yellow water as a substrate to obtain hexanoic acid.

[0017] 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 1:1, inoculating with the above-mentioned Clostridium krill SCP250302, or using the above-mentioned microbial agent, and then preparing hexanoic acid through fermentation.

[0018] In one embodiment of the present invention, the fermentation conditions are a temperature of 37°C, an inoculum size of 10% (v / v), and anaerobic static culture for 15 days.

[0019] The present invention also provides a method for hexanoic acid fermentation using the above-mentioned strain. In a composite fermentation system with simulated yellow water substrate, the glucose concentration is fixed at 6 g / L and the ethanol concentration is 3% (v / v). With the total carbon molar amount of lactic acid and acetic acid being constant (1.5 mol C / L) as a constraint, a full-proportion gradient of lactic acid:acetic acid carbon molar ratio of 1:0 to 0:1 is set. The strain is inoculated at an inoculum of 10% (v / v) and cultured anaerobically at 37°C for 15 days.

[0020] In one embodiment of the present invention, the strain achieves the highest hexanoic acid yield of 12.51 g / L under the condition of lactic acid:acetic acid = 1:1. Moreover, the hexanoic acid yield does not vary significantly within a wide range of lactic acid / acetic acid ratios, demonstrating a high degree of adaptability to substrate ratio fluctuations. Stable synthesis of hexanoic acid can be achieved without prior adjustment of the substrate ratio.

[0021] In one embodiment of the present invention, a variety of volatile flavor compounds are simultaneously accumulated in the fermentation system, including alcohols represented by n-hexanol and octanol, pyrazines represented by 2,5-dimethylpyrazine, and ester compounds represented by ethyl hexanoate, which significantly enriches the flavor composition of the fermentation product.

[0022] In one embodiment of the present invention, the strain has a strong ability to extend and derivatize carbon chains under lactic acid / acetic acid complex substrate conditions, and can synthesize high-carbon chain alcohol metabolites such as n-hexanol.

[0023] The present invention also provides the application of the above strains or the above microbial agents in the following scenarios: (1) resource-based hexanoic acid enhanced fermentation in yellow water systems with large substrate composition fluctuations or high acetic acid content; (2) targeted enhancement of hexanoic acid and flavor substances in the production of strong-aroma baijiu; (3) simplified hexanoic acid fermentation process without substrate pretreatment.

[0024] Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: (1) The hexanoic acid-producing strain provided by the present invention has typical acetic acid-preferred metabolic characteristics. In a single substrate system with acetic acid as the only carbon source, the hexanoic acid yield is the highest, reaching 6.46 g / L. This indicates that the strain has outstanding direct metabolic utilization ability of acetic acid and can efficiently convert acetic acid into hexanoic acid, providing a strain basis for the targeted conversion and utilization of abundant acetic acid resources in yellow water.

[0025] (2) The hexanoic acid-producing strain provided by this invention exhibits high adaptability to fluctuations in the lactic acid / acetic acid substrate ratio. It can maintain effective hexanoic acid production across the entire lactic acid:acetic acid ratio range from 1:0 to 0:1, with no significant differences in hexanoic acid yield among the treatment groups. In particular, the hexanoic acid yield reaches a peak of 12.51 g / L under the condition of lactic acid:acetic acid = 1:1. Compared with strains that require adjustment of the substrate ratio to a specific range for efficient acid production, this strain can be directly applied without prior adjustment of the substrate ratio in the fermentation system, significantly reducing dependence on the yellow water pretreatment process, simplifying the production operation process, and reducing production costs.

[0026] (3) The hexanoic acid-producing strain provided by the present invention has strong tolerance to ethanol and acetic acid. The above characteristics enable the strain to survive stably and maintain metabolic activity in the actual fermentation environment of yellow water, and have stronger industrial application adaptability.

[0027] (4) The optimal growth pH of the hexanoic acid-producing strain provided by the present invention is 5.0~6.5. It also has a wide range of carbon source utilization spectrum and can effectively utilize a variety of natural substrate components in yellow water for growth and metabolism. It has good overall adaptability to the complex substrate environment of yellow water.

[0028] (5) The hexanoic acid producing strain provided by the present invention can efficiently synthesize hexanoic acid in a complex substrate fermentation system, while simultaneously promoting the generation of various volatile flavor substances such as alcohols, pyrazines, and esters, significantly enriching the flavor composition of the fermentation products, and providing strain support for the targeted regulation of the flavor of strong-aroma baijiu.

[0029] Preservation of biological materials A strain of Clostridium krychnifolium Clostridium kluyverii SCP250302, taxonomic name is Clostridium Kluiver It was deposited on April 2, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 68031, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0030] Figure 1 The present invention is based on Clostridium kuristegia (Clostridium kuristegiae). Clostridium kluyveriiEthanol tolerance diagram of SCP250302.

[0031] Figure 2 The present invention is based on Clostridium kuristegia (Clostridium kuristegiae). Clostridium kluyverii Salt tolerance diagram of SCP250302.

[0032] Figure 3 The present invention is based on Clostridium kuristegia (Clostridium kuristegiae). Clostridium kluyverii Acetic acid tolerance diagram of SCP250302.

[0033] Figure 4 The present invention is based on Clostridium kuristegia (Clostridium kuristegiae). Clostridium kluyverii Hexanoic acid tolerance diagram of SCP250302. Detailed Implementation

[0034] 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.

[0035] The culture media involved in the following examples are as follows: 1 L of enhanced Clostridium tumefaciens medium (RCM) contains: 10 g peptone, 3 g yeast extract, 5 g glucose, 1 g soluble starch, 3 g sodium acetate, 0.5 g cysteine ​​hydrochloride, 20 g agar powder, and 1 mL of 0.1% (m / v) resazurin; pH = 6.28, sterilized at 115℃ for 30 min.

[0036] 1 L of sodium ethanolacetate medium contains: 6 g sodium acetate, 5 g yeast extract, 0.4 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.5 g ammonium sulfate, and 2% ethanol (added after sterilization). pH = 7.0, sterilized at 115℃ for 30 min.

[0037] 1 L 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). It is autoclaved at 115℃ for 30 min. The metal element stock solution (1 L) contains: 15 g ferrous sulfate, 10 g zinc sulfate, 10 g calcium chloride, and 10 g cobalt chloride.

[0038] Different carbon source media: The following single carbon sources were added to the basal medium: glucose, lactic acid, acetic acid, and ethanol. Using 12 g / L glucose as the carbon basis, other carbon sources were replaced by equal amounts based on their molar carbon content. The media were then autoclaved at 115°C for 30 min before use. Simulated yellow water culture medium: Lactic acid / acetic acid was added to the basal culture medium, and 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.

[0039] The detection methods involved in the following examples are as follows: Glucose detection methods: The glucose content was determined using the 3,5-dinitrosalicylic acid (DNS) colorimetric method, and the specific steps are as follows: (1) Preparation of standard curve: Accurately weigh 0.01 g of anhydrous glucose dried to constant weight at 105℃, prepare a standard stock solution with a concentration of 1.00 mg / mL, and serially dilute it to a series of standard solutions of 100~1000 µg / mL (interval of 100 µg / mL); take an appropriate amount of each, add 1.5 mL of DNS reagent, react in a boiling water bath for 15 min, cool and make up to 25 mL, measure the absorbance at a wavelength of 540 nm, and establish a standard curve with concentration as the abscissa and absorbance as the ordinate.

[0040] (2) Sample determination: Take 200 µL of sample solution, add 300 µL of DNS reagent, and mix thoroughly; react in a boiling water bath for 5 min, then rapidly cool to room temperature, and dilute with 1.5 mL of distilled water; measure OD at 540 nm. 540 The value is used to calculate the reducing sugar content in the sample based on the standard curve.

[0041] The detection methods for short / medium chain fatty acids and ethanol are as follows: The content of short / medium chain fatty acids (S / MCFAs) was determined by high performance liquid chromatography (HPLC). The specific procedure is as follows: (1) Sample pretreatment: Take 0.5 mL of fermentation broth (dilute appropriately if necessary), centrifuge at 12000 rpm for 2 min to remove the cells, take the supernatant, filter it through a 0.22 µm microporous membrane, and set it aside.

[0042] (2) Chromatographic conditions: The chromatograph was an Agilent 1260 Infinity II high performance liquid chromatograph (Agilent Technologies, USA); the chromatographic column was a Carbomix H column; the mobile phase was 2.5 mmol / L dilute sulfuric acid solution; the flow rate was 0.6 mL / min; and the column temperature was 55℃.

[0043] Volatile flavor compounds were detected using GC-MS, and the specific procedure is as follows: (1) Sample pretreatment: Centrifuge the fermentation broth at 8000 rpm for 10 min, take 3 mL of the supernatant and put it into a 20 mL brown headspace bottle, add 1 g of sodium chloride and 10 µL of chromatographic grade 2-octanol internal standard solution, and seal for later use.

[0044] (2) GC conditions: The instrument was a Shimadzu QP-2010SE gas chromatograph-mass spectrometer; the column was a VF-WAXms capillary column (60 m × 0.25 mm × 0.25 µm, Thermo Fisher Scientific, USA); the carrier gas was high-purity helium, and the injection was splitless; the injection port temperature was 250℃; the temperature program was as follows: hold at 40℃ for 2 min, increase the temperature to 130℃ at 3℃ / min, then increase the temperature to 200℃ at 6℃ / min, and finally increase the temperature to 230℃ at 8℃ / min and hold for 8 min.

[0045] (3) MS conditions: EI ionization source; ion source temperature 250℃; interface temperature 250℃; electron energy 70 eV; mass scan range 25~350 amu.

[0046] Example 1: Isolation and screening of Clostridium krillion The specific steps are as follows: Using the cellar mud from a strong-aroma baijiu brewing enterprise in Sichuan as experimental material, samples were immediately transferred and preserved under low-temperature anaerobic conditions after sampling.

[0047] (1) Enrichment culture The RCM liquid culture medium was dispensed into 150 mL capped glass bottles, with a volume of 80 mL per bottle. After autoclaving at 115℃ for 30 min, the bottles were placed in an anaerobic chamber for complete deoxygenation before use. 10 g of pit mud sample was weighed and directly inoculated into the above culture medium. The sample was gently shaken to ensure complete dispersion and then anaerobically cultured at 37℃ for 5 days. The resulting culture was used for subsequent separation operations.

[0048] (2) Dilution, coating and separation Take 1 mL of the enriched culture medium obtained in step (1) and add it to 9 mL of sterile physiological saline. Mix thoroughly and then perform a tenfold serial dilution to 10⁻⁶. -7 Take the dilution of 10... -3 10 -4 10 -5 10 -6 and 10 -7Two hundred µL of bacterial suspension were inoculated onto pre-prepared solid culture medium plates, spread evenly with a sterile spreader, and then incubated in an anaerobic incubator at 37 °C for 5–14 days. During the incubation period, the colony growth was observed regularly, and representative single colonies were selected based on colony morphology characteristics and purified by the streak plate method to obtain pure cultures.

[0049] (3) Liquid culture verification The purified strain was transferred to liquid culture medium and cultured under anaerobic conditions at 37°C for 3-5 days to obtain a single strain with stable growth.

[0050] (4) Strain identification and nomenclature Genomic DNA was extracted from the above strains, the 16S rRNA gene was amplified and analyzed by Sanger sequencing, and combined with the verification of hexanoic acid production ability, a hexanoic acid-producing strain was finally screened and named SCP250302.

[0051] 2. Molecular identification of Clostridium kuristeum Take 2 mL of the bacterial culture obtained from step 1, centrifuge at 8000 rpm for 5 min, collect the bacterial pellet, and extract genomic DNA using a bacterial genomic DNA extraction kit. The obtained DNA is used as a template for PCR amplification.

[0052] 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 conditions were as follows: pre-denaturation at 94℃ for 5 min; followed by 35 cycles, each cycle consisting of denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 90 s, and a final extension at 72℃ for 10 min. After verifying band size by 1% agarose gel electrophoresis, the PCR products were sent to Chengdu Sangon Biotech Co., Ltd. for Sanger sequencing.

[0053] The obtained 16S rRNA gene sequence was submitted to the NCBI database for BLAST homology comparison. The results showed that this strain was similar to Clostridium krillion (Clostridium krillion). Clostridium kluyverii The sequence similarity was 99.93%; combined with colony morphology observation and physiological and biochemical characteristic analysis, the strain was identified as Clostridium krillii and named SCP250302.

[0054] The 16S rDNA sequence is as follows (SEQ ID NO.3): Example 2: Performance determination of Clostridium krillion SCP250302 1. Acid resistance test (1) Preparation of culture media with different pH values The initial pH of the basal culture medium was adjusted to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively. Each pH was then adjusted to the target value using 5 mol / L HCl or NaOH solution. After sterilization, the pH deviation was controlled within ±0.1. The different pH media were aliquoted into 30 mL centrifuge tubes, with three biological replicates for each pH treatment.

[0055] The basic culture medium is as follows: 1 L 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). It is autoclaved at 115℃ for 30 min. The metal element stock solution (1 L) contains: 15 g ferrous sulfate, 10 g zinc sulfate, 10 g calcium chloride, and 10 g cobalt chloride.

[0056] (2) Preparation of seed liquid The *Clostridium krillion* SCP250302 strain obtained in Example 1, stored in cryovials at -80°C and 25% glycerol, was inoculated into 5 mL of sodium ethanolacetate medium and statically activated under anaerobic conditions at 37°C for 36 h to prepare a culture medium. Subsequently, at an inoculum rate of 10% (v / v), the prepared culture medium was transferred to 15 mL of sodium ethanolacetate medium for the first subculture, and anaerobically incubated at 37°C for 36 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase (i.e., after 36 h of incubation), it was transferred again at an inoculum rate of 10% (v / v) to 150 mL of sodium ethanolacetate medium for the second subculture, and anaerobically incubated at 37°C for 36 h. The resulting bacterial culture was the seed culture used in subsequent fermentation experiments.

[0057] (3) The activated Clostridium krill SCP250302 seed culture was inoculated into the culture medium 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.

[0058] OD of Clostridium krillion SCP250302 under different initial pH conditions 600The changes are shown in Table 1.

[0059] Table 1: OD under different pH conditions 600

[0060] The results showed that the strain could maintain a certain growth activity within the pH range of 4.0–9.0, and the OD... 600 A pH value between 0.11 and 0.22 indicates that this strain has a wide pH tolerance range. The strain grows most vigorously within the pH range of 5.0 to 6.5, with an OD value between 0.11 and 0.22. 600 All values ​​were greater than 0.16, higher than other pH conditions.

[0061] 2. Carbon source utilization spectrum (1) Preparation of culture medium In the basal medium without ethanol, glucose was replaced with arabinose (6.00 g / L), maltose (5.70 g / L), D-mannitol (6.07 g / L), fructose (6.00 g / L), sucrose (5.70 g / L), lactose (5.70 g / L), sodium pyruvate (7.34 g / L), ethanol (4.60 g / L), L-fucose (5.47 g / L), xylose (6.00 g / L), and soluble starch (5.40 g / L) (11 in total) as the sole added carbon source, while the other components of the medium remained unchanged. At the same time, the carbon source utilization of the strain was detected using the basal medium without ethanol (containing glucose) as the 12th carbon source medium.

[0062] The basal culture medium with ethanol removed is as follows: 1 L 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, and 6 g glucose. It is autoclaved at 115℃ for 30 min. The metal element stock solution (1 L) contains: 15 g ferrous sulfate, 10 g zinc sulfate, 10 g calcium chloride, and 10 g cobalt chloride.

[0063] 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.

[0064] (2) Preparation of seed liquid Clostridium krillion SCP250302, obtained in Example 1 and stored in cryovials at -80°C and 25% glycerol, was inoculated into 5 mL of sodium ethanolacetate medium and cultured anaerobically at 37°C for 36 h to prepare a culture medium. Subsequently, at a 10% (v / v) inoculation rate, the prepared culture medium was transferred to 15 mL of sodium ethanolacetate medium for the first subculture, and cultured anaerobically at 37°C for 36 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase (i.e., after 36 h of culture), it was transferred again at a 10% (v / v) inoculation rate to 150 mL of sodium ethanolacetate medium for the second subculture, and cultured anaerobically at 37°C for 36 h. The resulting bacterial culture was the seed culture used in subsequent fermentation experiments.

[0065] (3) The activated Clostridium krill SCP250302 seed culture was inoculated into the culture medium 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.

[0066] The growth of Clostridium krillion SCP250302 under various carbon source conditions is shown in Table 2.

[0067] Table 2: OD under different carbon source conditions 600

[0068] The results showed that the strains exhibited significant differences in their ability to utilize different carbon sources under 12 different conditions. The strains grew relatively well when using arabinose, fructose, lactose, ethanol, L-fucose, and glucose as carbon sources, with higher OD values. 600 The peak values ​​were 0.12, 0.15, 0.17, 0.11, 0.2 and 0.12, respectively, and the strains were able to grow normally.

[0069] Under the conditions of the other six carbon sources, including maltose, D-mannitol, sucrose, sodium pyruvate, xylose, and soluble starch, OD 600 The values ​​were all below 0.1, indicating extremely weak bacterial growth and limited ability of the strain to utilize the aforementioned carbon sources.

[0070] The above results indicate that the strain Clostridium krillion SCP250302 can utilize a variety of carbon sources, including arabinose, fructose, lactose, ethanol, L-fucose, and glucose, to grow, demonstrating a certain degree of broad carbon source utilization and good adaptability to common substrate components in yellow water.

[0071] 3. Ethanol tolerance (1) Preparation of culture media with different ethanol contents To evaluate the strain's tolerance to ethanol, an ethanol concentration gradient experimental system was constructed by adding different amounts of anhydrous ethanol (sterilized by organic membrane filtration) to a basal medium with ethanol components removed. The ethanol volume fractions for each experimental group were set to 0% (v / v), 2% (v / v), 4% (v / v), 6% (v / v), 8% (v / v), and 10% (v / v), respectively.

[0072] The basal culture medium with ethanol components removed is as follows: 1 L 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, and 6 g glucose. It is autoclaved at 115℃ for 30 min. The metal element stock solution (1 L) contains: 15 g ferrous sulfate, 10 g zinc sulfate, 10 g calcium chloride, and 10 g cobalt chloride.

[0073] (2) Preparation of seed liquid The *Clostridium krillion* SCP250302 strain obtained in Example 1, stored in cryovials at -80°C and 25% glycerol, was inoculated into 5 mL of sodium ethanolacetate medium and statically activated under anaerobic conditions at 37°C for 36 h to prepare a culture medium. Subsequently, at an inoculum rate of 10% (v / v), the prepared culture medium was transferred to 15 mL of sodium ethanolacetate medium for the first subculture, and anaerobically incubated at 37°C for 36 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase (i.e., after 36 h of incubation), it was transferred again at an inoculum rate of 10% (v / v) to 150 mL of sodium ethanolacetate medium for the second subculture, and anaerobically incubated at 37°C for 36 h. The resulting bacterial culture was the seed culture used in subsequent fermentation experiments.

[0074] (3) The seed liquid of Clostridium krusei SCP250302 was inoculated into the culture media with different ethanol concentrations obtained in step (1) above at an inoculation amount of 10% (v / v), and then placed in anaerobic conditions at 37°C for static culture for 72 h.

[0075] After the culture was completed, the OD of each group of fermentation broth was measured at a wavelength of 600 nm. 600 The effect of different ethanol concentrations on the growth of bacterial strains was investigated using bacterial cell optical density as an evaluation index.

[0076] The results are as follows Figure 1 As shown, the strains can achieve effective growth within the ethanol volume fraction range of 0% to 8% (v / v), indicating that they have strong ethanol tolerance.

[0077] Specifically, the strain exhibited good growth under conditions of 2% (v / v) to 4% (v / v) ethanol volume fraction, with OD... 600 The value can reach approximately 0.22~0.24; as the ethanol concentration increases to 6% (v / v) and above, the cell growth shows a gradual downward trend; even under conditions where the ethanol volume fraction is as high as 10% (v / v), it still has a certain growth capacity.

[0078] (4) Comparison of data The specific steps are the same as (1) to (3), the difference being that Clostridium krillion SCP250302 is adjusted as follows: Clostridium Kluiver DSM 555; Results show: In contrast, the OD of the same type of strain at an ethanol content of 6% (v / v) was... 600 A value of 0 indicates that its growth has been completely inhibited.

[0079] The strain described in this invention can still maintain a certain growth capacity at the same ethanol concentration (6% (v / v)).

[0080] This characteristic indicates that the Clostridium krillion SCP250302 strain has good tolerance and adaptability to high concentrations of ethanol, and has important potential application value in actual fermentation scenarios using complex substrates rich in ethanol, such as yellow water, as raw materials.

[0081] 4. Salt tolerance (1) Preparation of culture media with different sodium chloride contents Different amounts of sodium chloride were added to the basal culture medium to construct a salt concentration gradient experimental system. The sodium chloride mass concentrations of each experimental group were set to 2.5 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L and 25 g / L respectively.

[0082] The basic culture medium is as follows: 1 L 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). It is autoclaved at 115℃ for 30 min. The metal element stock solution (1 L) contains: 15 g ferrous sulfate, 10 g zinc sulfate, 10 g calcium chloride, and 10 g cobalt chloride.

[0083] (2) Preparation of seed liquid The *Clostridium krillion* SCP250302 strain obtained in Example 1, stored in cryovials at -80°C and 25% glycerol, was inoculated into 5 mL of sodium ethanolacetate medium and statically activated under anaerobic conditions at 37°C for 36 h to prepare a culture medium. Subsequently, at an inoculum rate of 10% (v / v), the prepared culture medium was transferred to 15 mL of sodium ethanolacetate medium for the first subculture, and anaerobically incubated at 37°C for 36 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase (i.e., after 36 h of incubation), it was transferred again at an inoculum rate of 10% (v / v) to 150 mL of sodium ethanolacetate medium for the second subculture, and anaerobically incubated at 37°C for 36 h. The resulting bacterial culture was the seed culture used in subsequent fermentation experiments.

[0084] (3) The activated seed liquid obtained in step (2) was inoculated into the culture media with different sodium chloride contents obtained in step (1) at an inoculation rate of 10% (v / v) and placed in anaerobic static culture at 37°C for 72 h.

[0085] After the culture was completed, the optical density (OD) of each group of fermentation broth was measured at a wavelength of 600 nm. 600 The bacterial cell growth status was used to evaluate the strain's tolerance to different salt concentrations.

[0086] The results are as follows Figure 2 As shown, under sodium chloride concentration stress, the strain as a whole exhibited a trend of gradually weakening growth with increasing salt concentration. As the sodium chloride concentration gradually increased to 20 g / L, the OD... 600 The concentration gradually decreased to approximately 0.08, indicating that the strain has a good adaptability to low-salt environments (able to tolerate at least 10 g / L sodium chloride), while high-salt conditions have a certain inhibitory effect on its growth.

[0087] 5. Tolerance to acetic acid (1) Preparation of culture media with different acetic acid contents Lactic acid was added to the basal culture medium, and acetic acid concentrations were set at 2 g / L, 5 g / L, 10 g / L, 20 g / L, 40 g / L, 60 g / L, and 80 g / L, respectively.

[0088] The basic culture medium is as follows: 1 L 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, 6 g glucose, 3% ethanol (v / v), and 350 µL of metal element stock solution. It is autoclaved at 115℃ for 30 min. Metal element stock solution (1 L): 15 g ferrous sulfate, 10 g zinc sulfate, 10 g calcium chloride, and 10 g cobalt chloride.

[0089] (2) Preparation of seed liquid The *Clostridium krillion* SCP250302 strain obtained in Example 1, stored in cryovials at -80°C and 25% glycerol, was inoculated into 5 mL of sodium ethanolacetate medium and statically activated under anaerobic conditions at 37°C for 36 h to prepare a culture medium. Subsequently, at an inoculum rate of 10% (v / v), the prepared culture medium was transferred to 15 mL of sodium ethanolacetate medium for the first subculture, and anaerobically incubated at 37°C for 36 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase (i.e., after 36 h of incubation), it was transferred again at an inoculum rate of 10% (v / v) to 150 mL of sodium ethanolacetate medium for the second subculture, and anaerobically incubated at 37°C for 36 h. The resulting bacterial culture was the seed culture used in subsequent fermentation experiments.

[0090] (3) The activated seed culture obtained in step (2) was inoculated into culture media with different acetic acid contents obtained in step (1) at an inoculation rate of 10% (v / v). The media were then anaerobically cultured at 37°C for 3 days. The OD value of the fermentation broth after culture was measured at a wavelength of 600 nm. The results are as follows: Figure 3 As shown.

[0091] The results show: from Figure 3 It can be seen that the strain exhibits good growth under low to moderate concentrations of acetic acid (2 g / L~20 g / L), with a high OD value. 600 The growth rate gradually increased with increasing acetic acid concentration, reaching a peak at 20 g / L (approximately 0.31); when the acetic acid concentration further increased to above 40 g / L, the cell growth rate decreased.

[0092] Even under high concentrations of acetic acid of 60 g / L and 80 g / L, the strain still showed a certain growth trend, indicating that high concentrations of acetic acid had a certain inhibitory effect on the growth of the strain, but the strain as a whole still had a relatively wide tolerance range for acetic acid concentration.

[0093] 6. Tolerance to hexanoic acid (1) Preparation of culture media with different hexanoic acid contents Add hexanoic acid to the basal culture medium and set the hexanoic acid concentrations as follows: 0, 2, 4, 6, 8, 10, and 12 (g / L).

[0094] The basic culture medium is as follows: 1 L 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). It is autoclaved at 115℃ for 30 min. The metal element stock solution (1 L) contains: 15 g ferrous sulfate, 10 g zinc sulfate, 10 g calcium chloride, and 10 g cobalt chloride.

[0095] (2) Preparation of seed liquid The *Clostridium krillion* SCP250302 strain obtained in Example 1, stored in cryovials at -80°C and 25% glycerol, was inoculated into 5 mL of sodium ethanolacetate medium and statically activated under anaerobic conditions at 37°C for 36 h to prepare a culture medium. Subsequently, at an inoculum rate of 10% (v / v), the prepared culture medium was transferred to 15 mL of sodium ethanolacetate medium for the first subculture, and anaerobically incubated at 37°C for 36 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase (i.e., after 36 h of incubation), it was transferred again at an inoculum rate of 10% (v / v) to 150 mL of sodium ethanolacetate medium for the second subculture, and anaerobically incubated at 37°C for 36 h. The resulting bacterial culture was the seed culture used in subsequent fermentation experiments.

[0096] (3) The Clostridium krusei SCP250302 seed liquid obtained in step (2) was inoculated into the culture medium with different hexanoic acid concentrations obtained in step (1) at an inoculation amount of 10% (v / v), and anaerobic static cultured at 37℃ for 3 days. The fermentation broth after culture was taken and its OD value was measured at a wavelength of 600 nm.

[0097] The results are as follows Figure 4 As shown.

[0098] The results show: Clostridium krill SCP250302 exhibits strong tolerance to hexanoic acid.

[0099] The strain exhibited good growth under low to moderate concentrations (0–4 g / L) of hexanoic acid, with an OD value of [missing value]. 600 The values ​​were all above 0.2, indicating that the strain had stable growth activity within this concentration range. When the hexanoic acid concentration was further increased to above 6 g / L, the OD... 600The value remained above 0.1, indicating that the strain as a whole still possessed a certain degree of hexanoic acid tolerance. However, when the hexanoic acid concentration further increased to above 8 g / L, the bacterial growth showed a decreasing trend.

[0100] This indicates that high concentrations of hexanoic acid have a limited inhibitory effect on the growth of Clostridium krillion SCP250302, and that Clostridium krillion SCP250302 has good tolerance to concentrations of hexanoic acid.

[0101] Example 3: Determination of hexanoic acid fermentation performance of Clostridium krillion SCP250302 under different carbon source conditions The specific steps are as follows: (1) Preparation of culture medium In the basal culture medium (excluding glucose), the following carbon sources were added: glucose, lactic acid, acetic acid, and ethanol (four in total). Using 12 g / L glucose as the carbon basis, the other carbon sources were replaced by an equal amount of carbon molarity. After conversion, the values ​​of lactic acid, acetic acid, and ethanol were 12 g / L and ethanol, respectively.

[0102] The basal culture medium without glucose is as follows: 1 L 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, and 3% ethanol (v / v). It is autoclaved at 115℃ for 30 min. The metal element stock solution (1 L) contains: 15 g ferrous sulfate, 10 g zinc sulfate, 10 g calcium chloride, and 10 g cobalt chloride.

[0103] 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.

[0104] (2) Preparation of seed liquid Clostridium krillion SCP250302, obtained in Example 1 and stored in cryovials at -80℃ and 25% glycerol, was inoculated into 5 mL of sodium ethanolacetate medium and cultured anaerobically at 37℃ for 36 h to prepare a culture medium. Subsequently, at a 10% (v / v) inoculation rate, the prepared culture medium was transferred to 15 mL of sodium ethanolacetate medium for the first subculture, and cultured anaerobically at 37℃ for 36 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase, it was transferred again at a 10% (v / v) inoculation rate to 150 mL of sodium ethanolacetate medium for the second subculture, and cultured anaerobically at 37℃ for 36 h. The resulting bacterial culture was the seed culture used in subsequent fermentation experiments.

[0105] (3) The Clostridium krill SCP250302 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, and samples were taken periodically. After fermentation, the content of short-chain and medium-chain fatty acids was determined by high performance liquid chromatography (HPLC).

[0106] The results are shown in Table 3 below: Table 3: Fermentation endpoints (12 days) for different carbon sources: substrate consumption, short- and medium-chain fatty acid accumulation, growth, and pH.

[0107] The results showed that the strain produced the highest hexanoic acid yield (6.46 g / L) when acetic acid was used as the sole carbon source, indicating that acetic acid was the dominant substrate for hexanoic acid synthesis by this strain, demonstrating its outstanding direct utilization ability of acetic acid. When glucose, lactic acid, and ethanol were used as carbon sources, the hexanoic acid yields were 0.72 g / L, 0.6 g / L, and 0.7 g / L, respectively. There were no significant differences among the three groups, but all yields were significantly lower than that of the acetic acid group.

[0108] The above results indicate that the strain exhibits typical acetic acid-biased metabolic characteristics, enabling it to efficiently drive hexanoic acid synthesis directly using acetic acid as a substrate, while its utilization of glucose, lactic acid, and ethanol is similar. Given that acetic acid is one of the main organic acid components in yellow water, this strain can directly utilize naturally occurring acetic acid in yellow water for hexanoic acid fermentation, thus possessing the substrate utilization basis for the resource-based production of hexanoic acid from yellow water.

[0109] Example 4: Effect of lactic acid / acetic acid substrate ratio on the fermentation performance of Clostridium krillion for hexanoic acid production The specific steps are as follows: (1) Preparation of culture medium 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 10 g / L (approximately 3%, v / v) in the basal medium to reflect the actual average levels of glucose and ethanol in the yellow water.

[0110] 1 L of 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); the metal element stock solution (1 L) contains: 15 g ferrous sulfate, 10 g zinc sulfate, 10 g calcium chloride, and 10 g cobalt chloride.

[0111] Based on this, lactic acid and acetic acid were added to the above culture medium. Using the total carbon molar amount of lactic acid and acetic acid as a constant (1.5 mol C / L) as a constraint, the relative ratio of lactic acid to acetic acid was adjusted to achieve single-variable control, resulting in a total of 7 treatment groups: 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.

[0112] 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.

[0113] 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.

[0114] (2) Preparation of seed liquid Clostridium krillion SCP250302, preserved in -80℃, 25% glycerol cryovials as obtained in Example 1, was inoculated into 5 mL of sodium ethanolacetate medium and statically activated under anaerobic conditions at 37℃ for 36 h to prepare a culture medium. Subsequently, at a 10% (v / v) inoculation rate, the prepared culture medium was transferred to 15 mL of sodium ethanolacetate medium for the first subculture, and anaerobically incubated at 37℃ for 36 h to restore and stabilize the strain's metabolic activity. After the bacterial culture reached the logarithmic growth phase (i.e., after 36 h of incubation), it was transferred again at a 10% (v / v) inoculation rate to 150 mL of sodium ethanolacetate medium for the second subculture, and anaerobically incubated at 37℃ for 36 h. The resulting bacterial culture was the seed culture used in subsequent fermentation experiments.

[0115] (3) The activated Clostridium kurlii seed culture was inoculated into fermentation media with different lactic acid / acetic acid ratios obtained in step (1) 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).

[0116] The substrate consumption and acid production results for each treatment group are shown in Table 4.

[0117] 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.

[0118] The results show: (1) In the pure lactic acid group (lactic acid:acetic acid = 1:0), the lactic acid consumption reached 4.61 g / L, and the hexanoic acid production at the end of fermentation was about 1.83 g / L, which was the lowest among the seven groups. The peak value of butyric acid at the same time was about 0.16 g / L, indicating that the hexanoic acid synthesis efficiency of the strain was relatively limited under the pure lactic acid substrate conditions.

[0119] (2) In the high lactic acid group, the lactic acid:acetic acid = 10:1 group and the 5:1 group had hexanoic acid production of 7.21 g / L and 8.22 g / L at the end of fermentation, respectively, which was higher than that of the pure lactic acid group. This indicates that under the substrate conditions where lactic acid is dominant, the introduction of a small amount of acetic acid has a certain promoting effect on the hexanoic acid synthesis of the strain.

[0120] (3) In the equal ratio group (lactic acid: acetic acid = 1:1), the hexanoic acid yield at the end of fermentation was about 12.51 g / L. The higher lactic acid group showed a significant increase, and it was the treatment group with the highest hexanoic acid yield among the seven groups. At the same time, butyric acid accumulated to about 9.83 g / L.

[0121] (4) In the high acetic acid group and the pure acetic acid group (lactic acid:acetic acid = 1:5, 1:10 and 0:1), as the proportion of acetic acid further increased, the hexanoic acid yield of each group was about 9 g / L, and there was no significant decrease, indicating that the strain has a certain tolerance and utilization ability to higher concentrations of acetic acid; however, the yield was lower than that of the equal proportion group, indicating that the excessively high proportion of acetic acid still has a certain inhibitory effect on hexanoic acid synthesis.

[0122] Furthermore, as the proportion of acetic acid increased, the accumulation of butyric acid also increased, reaching a maximum of 13.96 g / L, indicating that the proportion of carbon flowing to short-chain fatty acids was improved under high acetic acid conditions.

[0123] In summary, the hexanoic acid production of the strain exhibited a pattern of initial increase followed by stabilization with variations in the lactic acid / acetic acid ratio: the hexanoic acid production was lowest under pure lactic acid conditions, gradually increasing with a moderate increase in the acetic acid ratio, reaching a peak at a lactic acid:acetic acid ratio of 1:1; under high acetic acid conditions, the production decreased slightly, but the differences between groups were not significant, and the overall level remained high. These results indicate that the moderate introduction of acetic acid promotes hexanoic acid synthesis in the strain, and the synergistic substrate supply of lactic acid and acetic acid is key to driving efficient hexanoic acid synthesis. Unlike strains that are highly sensitive to substrate ratios and whose acid production is significantly inhibited under high acetic acid conditions, this strain can maintain effective hexanoic acid production over a wide range of lactic acid / acetic acid ratios, demonstrating good adaptability to substrate ratio fluctuations and exhibiting stronger robustness in practical production scenarios where the composition of the yellow water substrate varies significantly from batch to batch.

[0124] (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 = 1:1) which had the highest hexanoic acid yield. A total of 32 volatile flavor compounds were detected, demonstrating excellent metabolic diversity. These mainly included 13 alcohols, 3 aldehydes, 2 ketones, 8 pyrazines, and 6 esters. Typical compounds and their contents are shown in Table 5.

[0125] Table 5: Content of volatile aroma compounds produced by Clostridium krillion under lactic acid:acetic acid = 1:1 fermentation

[0126] The results show: Alcohols were the most abundant flavor components in the fermentation products of this group. Hexanol was the most abundant, with a concentration of 49.35 mg / L, exhibiting a typical fruity aroma; followed by octanol, with a concentration of 14.78 mg / L, possessing both oily and citrus aromas. Both are important high-carbon-chain alcohol flavor compounds in the fermentation system. The large accumulation of hexanol indicates that the strain has a strong ability for carbon chain elongation and derivatization under lactic / acetic acid complex substrate conditions.

[0127] Furthermore, this strain exhibits excellent pyrazine synthesis capabilities, producing a variety of pyrazine flavor compounds without the need for additional strains. Pyrazine compounds also accumulate significantly, with 2,5-dimethylpyrazine showing the highest concentration at 13.16 mg / L, possessing a rich chocolate and cream aroma and being an important roasted aroma component in the baijiu flavor system. Among ester compounds, ethyl hexanoate has the highest concentration at 1.15 mg / L, exhibiting tropical fruit aromas and being a core characteristic flavor compound of strong-aroma baijiu; its abundant formation is closely related to the continuous accumulation of hexanoic acid in the fermentation system.

[0128] The significant accumulation of these various volatile flavor compounds indicates that the strain can not only efficiently synthesize hexanoic acid in the lactic acid / acetic acid complex substrate system, but also simultaneously drive the generation of multiple flavor compounds such as multi-carbon chain alcohols, pyrazines, and esters, significantly enriching the flavor composition and aroma layers of the fermentation products. Applying this strain to the resource-based fermentation of yellow water is expected to enhance the overall flavor quality of the fermentation system while achieving hexanoic acid enhancement, providing strain support for the targeted regulation of strong-aroma baijiu flavor.

[0129] 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 Clostridium kuribda ( Clostridium kluyveri SCP250302, characterized in that, The Clostridium krillione strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 68031 and deposit date of April 2, 2026.

2. The Clostridium krillion SCP250302 according to claim 1, characterized in that, The *Clostridium kuriformis* SCP250302 grows in a pH range of 4.0–9.0; it utilizes one or more of arabinose, fructose, lactose, ethanol, L-fucose, and glucose as a carbon source; it tolerates 0–10% (v / v) ethanol; it tolerates 2.5–25 g / L salt concentrations; it tolerates 2–80 g / L acetic acid concentrations; and it tolerates 0–12 g / L hexanoic acid concentrations.

3. A microbial preparation containing Clostridium krill SCP250302 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 Clostridium krillion SCP250302, or a fermentation broth containing Clostridium krillion SCP250302; preferably, the live cells of Clostridium krillion SCP250302 are freeze-dried Clostridium krillion SCP250302 cells or immobilized Clostridium krillion SCP250302 cells.

5. The microbial agent according to claim 3 or 4, characterized in that, The viable cell number of Clostridium celti in the microbial inoculant reaches 1 x 10 8 CFU / g or 1 x 10 8 CFU / mL or above.

6. A method for simultaneously producing hexanoic acid and butyric acid, characterized in that, The method involves fermenting Clostridium krill SCP250302 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 Clostridium krillione SCP250302 as described in claim 1 or 2, or using any of the microbial agents described in claims 3 to 5, with yellow water as a 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 1:1, inoculating with Clostridium krill SCP250302 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 Clostridium krill SCP250302 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 strong-aroma baijiu cellar mud, the directional fermentation of yellow water to produce hexanoic acid, or the industrial production of hexanoic acid.

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