A method for preparing lactic acid using combined bacterial strains
By combining Bacillus subtilis and Lactobacillus pentosus strains and employing a phased oxygen supply strategy, the problem of efficient conversion of lignocellulose and lactic acid production in distiller's grains was solved, achieving efficient and low-cost lactic acid preparation, simplifying the process and increasing yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHENGDU UNIV
- Filing Date
- 2026-05-29
- Publication Date
- 2026-06-30
AI Technical Summary
Existing technologies struggle to efficiently convert distiller's grains into lactic acid without exogenous enzymes or complex pretreatment. Furthermore, conflicts exist between aerobic and anaerobic bacteria in terms of oxygen requirements and metabolic pathways, resulting in low lignocellulose degradation efficiency and poor lactic acid yield.
A combination strain of Bacillus subtilis KC1 and Lactiplantibacillus pentosus LB9 was used to carry out fermentation through a staged oxygen supply strategy, first aerobic and then anaerobic. The endogenous cellulase activity of KC1 was utilized, combined with the lactic acid fermentation ability of LB9, to achieve the enzymatic hydrolysis of lignocellulose and the synthesis of lactic acid.
Under conditions without exogenous enzymes and excipients, a cellulose degradation rate of 56.1% and a hemicellulose degradation rate of 68.4% were achieved in the baijiu lees, with a lactic acid yield of 3.816 g/L. This significantly improved the substrate conversion rate, simplified the process, and reduced costs.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial fermentation technology, and more specifically to a method for preparing lactic acid using a combination of bacterial strains. Background Technology
[0002] Distillers' grains, a major solid byproduct of baijiu (Chinese liquor) brewing, have an annual output exceeding 40 million tons. Rich in nutrients such as cellulose (19.7%) and hemicellulose (15.7%), they represent a highly promising lignocellulosic biomass resource. However, currently, distillers' grains are mostly used as low-value animal feed or directly landfilled, resulting in resource waste and potential environmental pollution. Therefore, achieving high-value utilization of distillers' grains has become a key issue for the green transformation of the brewing industry.
[0003] Lactic acid, as a key platform chemical, has wide applications in food, pharmaceuticals, and biodegradable materials (such as polylactic acid), and global market demand continues to grow. Currently, commercial lactic acid production mainly relies on food crops such as corn and sugarcane, which raises ethical and resource conflicts involving competition for food and land. Therefore, there is an urgent need to develop sustainable production pathways using non-food lignocellulosic waste as substrates.
[0004] In recent years, microbial fermentation technology has provided a new approach for the conversion of distiller's grains, but existing research mainly focuses on improving the feed nutritional value of distiller's grains through microbial fermentation. For example, Guo Suhuan et al. (Screening of Fermentation Strains for Baijiu Distillers' Grains, Feed Industry, Vol. 33, No. 15, 2012, hereinafter referred to as "Reference 1") disclosed a method using Lactobacillus acidophilus (… Lactobacillus acidophilus Y1, Brewing Yeast ( Brettanomyces custersianus Y2, Trichoderma viride ( Trichoderma viride Y3 and Bacillus subtilis ( Bacillus subtilis This paper reports a method for solid-state fermentation of baijiu (Chinese liquor) lees using four strains of bacteria (Y1+Y2+Y3+Y4). The method employed a staged oxygen supply approach (1-4 days aerobic, 5-10 days anaerobic) at 30°C, using a solid-state culture medium supplemented with exogenous adjuvants such as wheat bran, corn flour, and soybean meal. Under the conditions of the complete combination of the four strains (Y1+Y2+Y3+Y4), the crude protein content reached 95.31%, the crude fiber degradation rate was 50.90%, the cellulose degradation rate was 50.2%, the hemicellulose degradation rate was 41.2%, the cellulase activity was 317.38 U / g, and the xylanase activity was 89.71 U / g. In another study by the same authors (Research on Microbial Fermentation of Baijiu Lees, Feed Research, 2012, No. 9, hereinafter referred to as "Reference 2"), the screening results of 14 combinations of the same four strains were further disclosed, using viable cell count as the main indicator. The results showed that the viable cell count was higher during the aerobic stage.
[0005] The purpose of both Reference 1 and Reference 2 is to improve the nutritional value of baijiu (Chinese liquor) lees for feed, with the final product being feed ingredients. Evaluation indicators include nutritional parameters such as crude protein content, crude fiber degradation rate, and amino acid composition. However, the fermentation systems in Reference 1 and Reference 2 both rely on the addition of exogenous auxiliary materials such as wheat bran, corn flour, and soybean meal. Their schemes cannot achieve the technical problem of efficient degradation of lignocellulose and targeted synthesis of the target product under conditions without exogenous enzymes and nutrients.
[0006] Overall, the industrial application of microbial fermentation for the conversion of distiller's grains still faces the following core challenges:
[0007] Firstly, lignocellulose has a dense structure and high natural resistance to degradation. Traditional processes often require costly physical, chemical, or enzymatic pretreatment, significantly increasing energy consumption and economic costs. Although References 1 and 2 utilize the enzyme-producing ability of microorganisms, the addition of exogenous auxiliary materials in their solid-state fermentation systems objectively reduces the system's requirements for lignocellulose degradation efficiency, failing to demonstrate degradation efficiency in pure distiller's grains substrate.
[0008] Secondly, existing single-strain fermentation systems struggle to simultaneously achieve efficient cellulose degradation and targeted lactic acid synthesis. Cellulase-producing strains (such as Bacillus subtilis) are mostly aerobic, while lactic acid-producing bacteria (such as Lactobacillus) are strictly anaerobic. This fundamental conflict in oxygen requirements, metabolic pathways, and optimal environmental conditions leads to low substrate utilization and unsatisfactory lactic acid yields. While References 1 and 2 employ an "aerobic-then-anaerobic" oxygen supply sequence, their primary aim is to compare the impact of different time stages on the growth of various strains. They fail to recognize, and even less propose, how to address the metabolic coordination between aerobic enzyme-producing bacteria and anaerobic fermenting bacteria within the same system through precise control of oxygen supply strategies.
[0009] Third, although existing studies have attempted to construct co-culture systems to integrate saccharification and fermentation processes, metabolic inhibition or competition easily occurs between microbial communities in the absence of effective oxygen supply regulation strategies, making it difficult to achieve synergistic effects. For example, not every combination of any Bacillus subtilis strain and any Lactobacillus strain can coexist harmoniously; antagonistic effects between strains and their inhibitory effects on each other's growth and enzyme production activities are key bottlenecks restricting the efficiency of mixed culture systems. References 1 and 2 only confirmed the absence of antagonism among the four strains used using the cross-cross method, but did not conduct more in-depth studies on inter-strain metabolic interactions (such as cross-promotion experiments in cell-free supernatants, evaluation of enzyme activity effects, etc.), nor did they provide a method for establishing highly efficient synergistic microbial communities based on specific screening.
[0010] Fourth, most existing co-culture systems still require the addition of exogenous cellulase or excipients, failing to truly achieve the "one-step" high-efficiency conversion pursued by Consolidated Bioprocessing (CBP)—that is, in the same reactor, using waste as the sole carbon source, simultaneously completing the in-situ enzymatic hydrolysis of lignocellulose and the targeted synthesis of target chemicals without adding exogenous enzyme preparations or performing complex pretreatment.
[0011] Therefore, it is necessary to develop a novel co-cultivation technology that requires no exogenous enzymes or complex pretreatment, and can systematically coordinate aerobic saccharification and anaerobic fermentation processes through specific strain combinations and controlled oxygen supply strategies, so as to fully release the conversion potential of cellulose and hemicellulose in the distiller's grains and achieve efficient, low-cost, and sustainable production of lactic acid. Summary of the Invention
[0012] This invention provides a method for preparing lactic acid using a combination of bacterial strains, aiming to solve the problem of efficient conversion of lignocellulose distiller's grains into lactic acid by constructing an efficient synthetic microbial community and an innovative oxygen supply strategy.
[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0014] A method for preparing lactic acid using a combination of bacterial strains includes the following steps:
[0015] (1) Prepare raw materials and microbial strains. The raw materials are lees from baijiu (Chinese liquor); the microbial strains include Bacillus subtilis. Bacillus subtilis KC1 and Lactobacillus pentosaccharide Lactiplantibacillus pentosus LB9, of which Bacillus subtilis Bacillus subtilis KC1 was deposited on December 11, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 67463), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. (Lactobacillus pentosus) Lactiplantibacillus pentosus LB9 was deposited on November 17, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67311, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0016] (2) Bacillus subtilis Bacillus subtilis KC1 and Lactobacillus pentosaccharide Lactiplantibacillus pentosusAfter activation, LB9 strains were mixed to form a composite strain, which was then inoculated into a culture medium using baijiu lees as the sole fermentation substrate without the addition of exogenous enzymes or exogenous excipients. During fermentation, a staged oxygen supply strategy was adopted: aerobic fermentation was carried out for 1–4 days, followed by anaerobic fermentation for 5–9 days. The volume ratio of Bacillus subtilis to Lactobacillus pentosus was 1–3:3–1, and the inoculation amount was 6%–14% of the fermentation medium volume. The amount of baijiu lees added to the fermentation medium was 10 g / L–60 g / L. The initial pH of the fermentation medium was 4.36–6.36.
[0017] (3) Collect lactic acid from fermentation products.
[0018] Preferably, the baijiu lees are lignocellulose baijiu lees.
[0019] Furthermore, in step (2), aerobic fermentation is carried out for 2 days first, followed by anaerobic fermentation for 5 days.
[0020] Preferably, in step (2), the Bacillus subtilis Bacillus subtilis KC1 and Lactobacillus pentosaccharide Lactiplantibacillus pentosus The volume ratio of LB9 was 1:1, and the inoculum volume was 11% of the fermentation medium volume.
[0021] Preferably, in step (2), the initial pH of fermentation is 5.64.
[0022] Preferably, in step (2), the amount of baijiu lees added to the fermentation culture medium is 10 g / L.
[0023] Furthermore, in step (2), before fermentation, Bacillus subtilis is first cultured in LB medium with a pH of 7.0. Bacillus subtilis KC1 is used for activation; the LB medium consists of: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and distilled water to a final volume of 1000 mL.
[0024] Furthermore, in step (2), before fermentation, Lactobacillus pentosus is first cultured on MRS medium. Lactiplantibacillus pentosus LB9 was activated; the composition of the MRS medium included: 10.0 g peptone, 10.0 g beef extract, 20.0 g glucose, 5.0 g yeast extract, 3.02 g sodium acetate trihydrate, 1.16 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate, 2.0 g triammonium citrate, 0.03 g manganese sulfate monohydrate, and 1.0 mL Tween 80, with distilled water added to bring the volume to 1000 mL.
[0025] Preferably, the fermentation temperature is 37°C and the rotation speed is 120 r / min.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] (1) This invention realizes the targeted production of lactic acid using baijiu lees as raw material and CBP combined bioprocessing, fundamentally breaking through the single technical route of existing technologies that only use lees for feed. References 1 and 2 both aim to improve the nutritional value of baijiu lees for feed, and the evaluation indicators are limited to feed nutrition parameters such as crude protein content and crude fiber degradation rate. They do not provide any guidance or inspiration for the production of platform chemicals such as lactic acid using lees as substrate. This invention uses the massive by-product of the baijiu brewing industry—baijiu lees—as the sole carbon source and sole nutrient substrate to produce high-value lactic acid, completely avoiding the dependence of the traditional lactic acid industry on grain crops such as corn and sugarcane, and alleviating the ethical and resource conflicts of "competing with people for food and with food for land". This technical path not only reduces the consumption of agricultural resources, but also effectively solves the resource waste and environmental pollution problems caused by brewing by-products being mostly used as low-value feed or directly landfilled. It not only reduces the production cost of lactic acid, but also constructs an efficient circular economy chain of "brewing-biochemical industry", realizing the resource utilization and value-added utilization of waste.
[0028] (2) The Bacillus subtilis strain constructed in this invention Bacillus subtilis KC1 and Lactobacillus pentosaccharide Lactiplantibacillus pentosus The synthetic microbial community composed of LB9 integrates the enzymatic hydrolysis and saccharification of lignocellulose with the anaerobic fermentation of lactic acid in a single reactor, using baijiu lees as the sole fermentation substrate without any exogenous auxiliary materials, thus achieving co-bioprocessing (CBP). In contrast, the fermentation systems in References 1 and 2 require the addition of exogenous auxiliary materials such as wheat bran, corn flour, and soybean meal. The addition of these auxiliary materials objectively provides readily available carbon and nitrogen sources, reducing the system's requirements for lignocellulose degradation capacity. The system of this invention fully utilizes the endogenous cellulase and xylanase synthesis capabilities of the preserved strain KC1—experiments have confirmed that KC1 can efficiently secrete endoglucanase (EG), β-glucosidase (BGL), and xylanase, eliminating the need for the addition of expensive commercial enzyme preparations. It also avoids high-energy-consuming and high-cost physical or chemical pretreatment of raw materials, greatly simplifying the operation process and reducing equipment investment and operating energy consumption. Under conditions without exogenous enzymes and without pretreatment, the cellulose degradation rate reached 56.1% and the hemicellulose degradation rate reached 68.4%, both of which are superior to the 50.2% cellulose degradation rate and 41.2% hemicellulose degradation rate obtained in Reference 1 under the condition of adding exogenous excipients. This fully demonstrates the significant progress of the CBP system of this invention in pure waste matrix compared with the existing technology, and provides a new technical path for low-cost and high-efficiency biorefining of lignocellulosic waste, which has the potential for industrial application.
[0029] (3) This invention screened LB9 strain from four candidate lactobacilli using the Oxford cup method, which showed no metabolic antagonism with KC1. Further, through cross-addition experiments with cell-free supernatants, it was confirmed that the cell-free supernatant of the preserved strain LB9 significantly promoted the growth of KC1, exhibiting the most prominent growth-promoting effect among all candidate lactobacilli. The cell-free supernatant of the preserved strain KC1 also significantly increased the biomass of LB9. More importantly, LB9 had the least inhibitory effect on the total cellulase activity of KC1 among all candidate lactobacilli, and all candidate lactobacilli had minimal impact on the xylanase activity of KC1. This specific bidirectional metabolic mutualistic relationship between the preserved strain KC1 and LB9 allows the lees from the liquor to simultaneously serve as a substrate for inducing enzyme production and a substrate for lactic acid fermentation, meeting all the nutritional needs of both strains. This enables the efficient completion of the entire fermentation process without the addition of any exogenous auxiliary materials such as bran, corn flour, soybean meal, or urea. Compared with existing technologies that require the addition of exogenous nutrients such as bran for solid-state fermentation, this method is not only simpler in terms of process, but also demonstrates the unique synergistic advantages of the combination of the preserved strains LB9 and KC1 in this invention.
[0030] (4) In response to the fundamental contradiction in oxygen demand between the cellulase-producing aerobic bacterium KC1 and the strictly anaerobic lactic acid fermenting bacterium LB9, this invention proposes a phased dynamic oxygen supply strategy of "aerobic first, then anaerobic". It should be noted that although Reference 1 and Reference 2 also arranged the time sequence of "aerobic 1-4 days, anaerobic 5-10 days" in their experimental design, their purpose was only to compare the differences in the growth of various strains under different oxygen supply conditions (Reference 2 used the number of viable bacteria as the only indicator to conclude that "the number of viable bacteria is higher in the aerobic stage"). They did not realize the fundamental conflict in metabolic pathways between aerobic enzyme-producing bacteria and anaerobic lactic acid fermenting bacteria, nor did they take the switching of oxygen supply time sequence as the core regulatory means to solve this conflict. In the aerobic stage, this invention fully mobilizes the enzyme production activity of Bacillus subtilis KC1, efficiently expresses cellulase and xylanase, and degrades cellulose and hemicellulose in the waste into fermentable sugars; in the anaerobic stage, it optimizes the fermentation metabolism of Lactobacillus pentosus LB9, and converts sugars into lactic acid through homolactic fermentation. Experimental data show that under static anaerobic conditions, the lactic acid yield of LB9+KC1 co-cultured for 7 days was only 1.678 g / L; however, after adopting a phased strategy of "2 days of aerobic + 5 days of anaerobic", the lactic acid yield significantly increased to 3.816 g / L, an increase of 127.33%, which fundamentally reconciled the contradiction between the microenvironment of enzymatic hydrolysis and saccharification and fermentation, and broke through the core bottleneck in the mixed culture system.
[0031] (5) Under optimized fermentation conditions (initial pH=5.64, total inoculum 11%, KC1 to LB9 volume ratio 1:1, fermentation temperature 37℃, fermentation period 6.5 days), the degradation rates of cellulose and hemicellulose in the baijiu lees of this invention reached 56.1% and 68.4%, respectively, and the lactic acid yield reached 3.816 g / L, corresponding to a substrate conversion rate of 0.382 g lactic acid / g dry lees. Scanning electron microscopy (SEM) observation further confirmed that after 6.5 days of fermentation, the surface of the lees changed from a dense and flat structure before fermentation to one full of pores, cracks and erosion marks, with a large number of microbial cells and filamentous biofilm structures attached, which directly reflects the efficient degradation and utilization of the lignocellulose matrix of the lees by the preserved strain combination. This invention combines single-factor experiments with Box-Behnken response surface methodology to systematically optimize and model key parameters in the co-culture system. The established regression model has high prediction accuracy with a relative error of only 0.95%. The determined process conditions are scientific, reliable, and highly repeatable, providing experimental support for the large-scale production of this technology.
[0032] (6) Each step of this invention is interconnected and mutually reinforcing (including the unique synergy of strains LB9 and KC1, the selection of substrates in the culture medium, and the design of various parameters). It not only provides a method for the bio-production of lactic acid using baijiu lees as the sole substrate, without the need for exogenous enzymes, exogenous excipients, or complex pretreatment, but also demonstrates a general technical blueprint for converting agricultural processing waste into high-value platform chemicals, namely: "no excipient addition—liquid deep fermentation—CBP—platform chemical production". This path conversion is a systematic creation based on the specific screened preserved strains KC1 and LB9 as carriers, the systematic resolution of metabolic conflicts as the core, and the phased oxygen supply as the control method. Moreover, this system is easy to integrate into the existing baijiu brewing industry, forming a green industrial chain of "distillery lees—lactic acid—polylactic acid", with high added value, significant emission reduction effect, and broad market prospects. Attached Figure Description
[0033] Figure 1 The figure shows the results of the antagonistic interaction between Bacillus subtilis KC1 and different lactic acid bacteria (LB5, JD11, D-1, LB9) on MRS solid medium by the Oxford cup method in this embodiment of the invention. A is the LB5 / KC1 combination, B is the JD11 / KC1 combination, C is the D-1 / KC1 combination, and D is the LB9 / KC1 combination.
[0034] Figure 2 This is a graph showing the effect of Lactobacillus cell-free supernatant on Bacillus subtilis KC1 biomass (OD600) in an embodiment of the present invention;
[0035] Figure 3This is a graph showing the effect of Bacillus subtilis KC1 cell-free supernatant on Lactobacillus biomass (OD600) in an embodiment of the present invention.
[0036] Figure 4 The figure shows the effect of Lactobacillus cell-free supernatant on the cellulase activity of Bacillus subtilis KC1 in the embodiments of the present invention, including endoglucanase (EG), β-glucosidase (BGL) and total cellulase activity.
[0037] Figure 5 This is a graph showing the effect of Lactobacillus cell-free supernatant on the xylanase activity of Bacillus subtilis KC1 in an embodiment of the present invention;
[0038] Figure 6 This is a dynamic pH change diagram of different bacterial strain combinations (LB5+KC1, JD11+KC1, D-1+KC1, LB9+KC1) during fermentation in the embodiments of the present invention.
[0039] Figure 7 This is a comparison chart of lactic acid production under different fermentation modes (single-strain, mixed-strain static anaerobic, and mixed-strain staged oxygen supply) in the embodiments of the present invention.
[0040] Figure 8 This is a graph showing the results of a single-factor experiment on the effect of inoculum size (6%–14%) on lactic acid production in an embodiment of the present invention.
[0041] Figure 9 This is a graph showing the results of a single-factor experiment on the effect of the concentration of discarded lees (10-60 g / L) on lactic acid production in an embodiment of the present invention.
[0042] Figure 10 This is a graph showing the results of a single-factor experiment on the effect of the inoculum volume ratio of Bacillus subtilis KC1 to Lactobacillus pentosus LB9 (1:3 to 3:1) on lactic acid production in an embodiment of the present invention.
[0043] Figure 11 This is a graph showing the results of a single-factor experiment on the effect of aerobic oxygen supply duration (1-5 days) on lactic acid production in an embodiment of the present invention.
[0044] Figure 12 This is a graph showing the results of a single-factor experiment on the effect of initial pH (4.36–6.36) on lactic acid production in an embodiment of the present invention.
[0045] Figure 13 This is a graph showing the results of a single-factor experiment on the effect of fermentation time (5-13 days) on lactic acid yield in an embodiment of the present invention.
[0046] Figure 14 The graph shows the monitoring results of acetic acid production under each single-factor experimental condition in the embodiments of the present invention.
[0047] Figure 15This is a three-dimensional response surface analysis diagram of the prediction model obtained by optimizing pH and fermentation time based on the Box-Behnken response surface methodology in an embodiment of the present invention.
[0048] Figure 16 This is a three-dimensional response surface analysis diagram of the prediction model obtained by optimizing pH and inoculum size based on the Box-Behnken response surface methodology in an embodiment of the present invention.
[0049] Figure 17 This is a three-dimensional response surface analysis diagram of the prediction model obtained by optimizing inoculum size and fermentation time based on the Box-Behnken response surface methodology in an embodiment of the present invention.
[0050] Figure 18 The images shown are microscopic morphology images of the discarded lees of Baijiu (Chinese liquor) observed by scanning electron microscopy (SEM) in an embodiment of the present invention. A represents the surface structure of the discarded lees before fermentation, while B, C, and D represent the surface structure of the discarded lees after 6.5 days of co-fermentation of LB9 and KC1. Detailed Implementation
[0051] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments.
[0052] Example
[0053] This embodiment provides a method using Bacillus subtilis. Bacillus subtilis KC1 and Lactobacillus pentosaccharide Lactiplantibacillus pentosus The Consolidated Bioprocessing (CBP) co-culture system constructed by LB9 is specifically designed for the efficient production of lactic acid from baijiu (Chinese liquor) lees. Among its components is Bacillus subtilis. Bacillus subtilis KC1 (hereinafter referred to as "KC1") has the accession number GDMCC NO: 67463; Lactobacillus pentosus Lactiplantibacillus pentosus The accession number of LB9 (hereinafter referred to as "LB9") is GDMCC NO: 67311. In this embodiment, using baijiu lees as the sole carbon source, a co-culture system was constructed using KC1 and LB9 to simultaneously complete the in-situ enzymatic degradation of lignocellulose and the directional synthesis of lactic acid in a single reactor, realizing the biotransformation from non-grain waste into high-value-added chemicals.
[0054] The design principles, construction basis, and specific preparation process of this embodiment will be systematically explained below.
[0055] In addition to KC1 and LB9, this embodiment also involves *Lactobacillus plantarum*. Lactiplantibacillus plantarum JD11 (hereinafter referred to as "JD11"), Lactobacillus plantarum Lactiplantibacillus plantarumLB5 (hereinafter referred to as "LB5"), Lactobacillus paracasei Lacticaseibacillus paracasei D-1 (hereinafter referred to as "D-1"), of which LB5 strain was obtained from the old mother water of radish kimchi, JD11 strain was obtained from the old mother water of cowpea kimchi, and D-1 strain was obtained from the accumulated waste residue. The screening process is as follows:
[0056] In a sterile operating room, 10.0 g of pickled vegetable brine, liquor cellar mud, and discarded lees samples were weighed and inoculated into Erlenmeyer flasks containing 90 mL of MRS liquid. The flasks were then incubated at 37°C for 24 h for enrichment. After enrichment, 1.0 mL of the culture was serially diluted (10⁻¹ to 10⁻⁶) using sterile physiological saline. 100 μL of bacterial solutions at dilutions of 10⁻⁴, 10⁻⁵, and 10⁻⁶ were spread onto MRS solid selection medium plates supplemented with 1.0% (w / v) CaCO₃ and incubated upside down at 37°C for 48 h. Colonies with clear zones were selected for isolation and purification. The purified strains were then inoculated into MRS seed culture medium for activation. Subsequently, the activated seed culture was inoculated at a 2% (v / v) inoculation rate into Erlenmeyer flasks containing 50 mL of MRS liquid medium and incubated at 37°C for 48 h for fermentation. After fermentation, the fermentation broth was centrifuged at 4℃ and 8000× g for 10 min to remove bacterial cells and macromolecular impurities.
[0057] The supernatant was collected, degassed by sonication for 30 min to remove dissolved gases, and then filtered through a 0.22 μm aqueous microporous membrane for later use. The lactic acid concentration of the filtrate was determined by high-performance liquid chromatography (HPLC), with qualitative analysis based on retention time and quantification using the external standard method. The conversion rate from substrate to lactic acid was calculated, and strains with a conversion rate of 80% or higher were classified as homofermenting lactic acid bacteria. HPLC conditions: column (Agilent ZORBAX SB-Aq-C18, 4.6 mm × 250 mm, 5 μm), mobile phase: 0.1% phosphoric acid-methanol (97.5:2.5), flow rate: 1.0 mL / min, detection wavelength: 210 nm, column temperature: 40℃, injection volume: 10 μL. LB5 and JD11 strains were obtained from old kimchi mother water. Strain D-1 was obtained from discarded lees. The MRS medium seed culture consists of: 10 g peptone, 10 g beef extract, 20 g glucose, 5 g yeast extract, 3.02 g sodium acetate trihydrate, 1.16 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate, 2 g triammonium citrate, 0.03 g manganese sulfate monohydrate, and 1.0 mL Tween 80, diluted to 1000 mL with distilled water.
[0058] The first step was preliminary screening for inter-strain compatibility and synergistic effects. The Oxford cup method was used to test the antagonism between KC1 and four candidate lactobacilli (LB5, JD11, D-1, and LB9). Specifically, each lactobacillus was inoculated into MRS liquid medium (formula: 10.0 g peptone, 10.0 g beef extract, 20.0 g glucose, 5.0 g yeast extract, 3.02 g CH3COONa·3H2O, 1.16 g K2HPO4, 0.05 g MgSO4, 2.0 g triammonium citrate, 0.03 g MnSO4·H2O, 1.0 mL Tween 80, and distilled water to a final volume of 1000 mL; sterilized at 121℃ for 15 min). The medium was then incubated statically at 37℃ for 24 h. The diluted culture was then evenly spread onto MRS solid plates as an indicator layer. After placing sterile Oxford cups, add an appropriate amount of diluted KC1 bacterial solution and incubate at 37°C for 24 hours. Observe the formation of inhibition zones. The results show that clear inhibition zones appear when LB5 and KC1 are co-cultured. Figure 1 A) indicates a clear metabolic antagonism between the two strains; conversely, no inhibition zone was observed when LB9 and KC1 were co-cultured. Figure 1 D), indicating that the two have good symbiotic compatibility. Figure 1 B), D-1 and KC1 ( Figure 1 C) No obvious inhibition zone was observed during co-culture, indicating that these three lactic acid bacteria have good compatibility with KC1 at a certain concentration.
[0059] To further investigate the interaction mechanisms among the strains, a cross-addition experiment of cell-free supernatant was conducted. Fermentation broth of KC1 cultured for 24 h in LB medium (formulation: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 1000 mL distilled water, pH 7.0, autoclaved at 121℃ for 20 min) and fermentation broth of each Lactobacillus cultured for 24 h in MRS medium were collected. Cell-free supernatant was obtained by filtration through a 0.22 μm filter membrane. The cell-free supernatant of Lactobacillus was added to the LB medium of KC1, with the unadded group serving as a control. After incubation at 37℃ and 120 rpm for 24 h with shaking, the OD600 value was measured. Results are as follows: Figure 2 As shown, except for LB5, cell-free supernatants from JD11, D-1, and LB9 significantly promoted the growth of KC1, with LB9 showing the most significant effect. In the reverse experiment, adding cell-free supernatants from KC1 to the MRS medium of each lactobacillus significantly increased the biomass of all lactobacilli compared to the control. Figure 3This indicates that the two bacteria can mutually promote each other's growth through the secretion of metabolites. Furthermore, by measuring cellulase activity, it was found that after adding cell-free supernatant from lactic acid bacteria, LB9 and JD11 promoted the activity of endoglucanase (EG) in KC1, while all lactic acid bacteria, except LB9, inhibited the activity of β-glucosidase (BGL) in KC1. Regarding total cellulase activity, all lactic acid bacteria fermentation broths reduced the total cellulase activity of KC1, with LB9 showing the least reduction. Figure 4 The xylanase activity assay results showed that the cell-free supernatant from all lactic acid bacteria had minimal effect on the xylanase activity of KC1. Figure 5 These results further demonstrate, at the enzymatic level, the significant synergistic potential of LB9 and KC1 in substrate degradation.
[0060] Based on the above results, LB9 and KC1 were ultimately selected to construct the CBP co-culture system. To address the conflict between oxygen demand in aerobic enzyme production and anaerobic fermentation, this embodiment innovatively proposes a staged oxygen supply strategy. The specific operation steps are as follows:
[0061] S1, Raw material pretreatment: Fresh lees from a strong-aroma baijiu enterprise in Sichuan Province were taken, the main components of which were rice husks and sorghum residue. The lees were dried in a 62℃ oven to constant weight, processed by a pulverizer and passed through a 60-mesh sieve. A basic salt solution (containing CH3COONa·3H2O 5 g / L, K2HPO4 2 g / L, MgSO4 0.2 g / L, triammonium citrate 1.4 g / L, MnSO4·H2O 0.05 g / L, CaCl2 0.1 g / L) was added at a concentration of 10 g / L to adjust the initial pH to 5.64. The mixture was then autoclaved at 121℃ for 15 min to obtain the fermentation medium.
[0062] S2, Strain Activation and Inoculation: KC1 was inoculated into LB liquid medium and activated by shaking at 37°C and 180 rpm for 12 h; LB9 was inoculated into MRS liquid medium and activated by static incubation at 37°C for 24 h. The activated KC1 and LB9 bacterial cultures were mixed at a 1:1 volume ratio to make a total inoculation amount of 11% (v / v) and then inoculated into sterilized fermentation medium.
[0063] S3, Aerobic Enzyme Production Stage: The inoculated system was placed in a shaker at 37℃ and 120 rpm for 2 days. During this stage, KC1 fully utilized oxygen to efficiently express endoglucanase (EG), β-glucosidase (BGL), and xylanase, initiating the hydrolysis of cellulose and hemicellulose in the slag, releasing fermentable sugars such as glucose and xylose.
[0064] S4, Anaerobic Fermentation Stage: After 2 days, stop shaking, seal the fermentation container and transfer it to static anaerobic conditions. Continue to cultivate at 37°C for 5 days. During this stage, LB9 converts sugars into lactic acid through homolactic acid fermentation. The total fermentation cycle is 6.5 days.
[0065] pH dynamics during fermentation of different strain combinations, such as Figure 6 As shown, after 7 days of fermentation, the pH values of all four combinations (LB5+KC1, JD11+KC1, D-1+KC1, LB9+KC1) tended to stabilize. Lactic acid yield results are as follows... Figure 7 As shown, under static anaerobic conditions, the lactic acid yield of co-cultured with KC1 by the four lactic acid bacteria strains was higher than that of single-strain fermentation, but still did not reach the ideal level. Under static anaerobic conditions throughout the entire process, the lactic acid yield of LB9+KC1 co-cultured for 7 days was only 1.678 g / L. However, after adopting a phased strategy of "2 days of aerobic + 5 days of anaerobic", the lactic acid yield of the LB9+KC1 combination significantly increased to 3.816 g / L, an increase of 127.33%. D-1+KC1, JD11+KC1, and LB5+KC1 showed a moderate degree of improvement. The above results demonstrate that this strategy effectively coordinates the physiological metabolic needs of the two strains.
[0066] After fermentation, the fermentation broth was centrifuged at 8000 × g / min for 10 min, and the supernatant was collected. The supernatant was then degassed by sonication for 30 min and filtered through a 0.22 μm microporous membrane. The lactic acid content was determined by high-performance liquid chromatography (HPLC). Chromatographic conditions: Agilent ZORBAX SB-Aq-C18 column (4.6 mm × 250 mm, 5 μm), mobile phase: 0.1% phosphoric acid-methanol (97.5:2.5, v / v), flow rate: 1.0 mL / min, detection wavelength: 210 nm, column temperature: 40℃, injection volume: 10 μL. The final lactic acid yield was 3.816 g / L, corresponding to a substrate conversion rate of 0.382 g lactic acid / g dry basis precipitate.
[0067] To further improve lactic acid production, this embodiment systematically optimized key process parameters through single-factor experiments. Initial conditions were set as follows: natural pH, temperature 37℃, rotation speed 120 rpm, inoculum size 10%, inoculum ratio 1:1, and slag concentration 40 g / L. The effects of each factor were investigated individually.
[0068] like Figure 8 As shown, as the inoculation amount increased from 6% to 10%, the lactic acid yield increased from 6.17 g / L to 7.57 g / L; when it was further increased to 14%, the yield tended to stabilize, so 10% was chosen as the central inoculation level.
[0069] From the perspective of the influence of substrate concentration ( Figure 9The highest lactic acid yield (7.57 g / L) was observed at a slag concentration of 40 g / L, but the conversion rate continuously decreased with increasing concentration, dropping from 41.54%. Considering both the inhibitory effect and cost factors, 10 g / L was ultimately selected as the optimal concentration for further optimization.
[0070] Results of vaccination ratio trial ( Figure 10 The results showed that when KC1 and LB9 were inoculated at a 1:1 ratio, both lactic acid yield and conversion rate reached their highest levels (8.01 g / L and 20.04%, respectively), while all other ratios showed a decrease. As the LB9 inoculation ratio increased, lactic acid yield began to decrease; similarly, as the KC1 inoculation ratio increased, lactic acid yield also showed a decreasing trend. Therefore, a 1:1 ratio was determined to be the optimal inoculation ratio.
[0071] The effect of aerobic time, such as Figure 11 As shown, lactic acid production is relatively stable under oxygen supply for 1 to 4 days; after 5 days, it decreases significantly due to the simultaneous decrease in lactic acid content and conversion rate. This may be because excessive oxygen supply inhibits the anaerobic fermentation metabolic pathway of lactic acid bacteria. Therefore, the duration of the aerobic stage is determined to be 2 days.
[0072] Initial pH optimization results show that ( Figure 12 Lactic acid production initially increased and then decreased with increasing pH, reaching a peak of 7.30 g / L (conversion rate 18.27%) at pH 5.36. This was attributed to the synergistic effect between the optimal pH range of lactic acid bacteria (5.0–6.0) and the pH range of enzyme activity of Bacillus subtilis KC1 (5.5–7.0). Therefore, the optimized pH range was set at 4.86–5.86.
[0073] The effect of fermentation time, such as Figure 13 As shown, the lactic acid yield and conversion rate were relatively high at 7 days (7.47 g / L and 18.68%, respectively). Extending the fermentation time actually led to a decrease in yield, but the lactic acid content and conversion rate rebounded to some extent at 13 days.
[0074] Meanwhile, the yield of acetic acid under each single-factor experimental condition was monitored, and the results are as follows: Figure 14 As shown.
[0075] Based on the results of the single-factor experiments above, the Box-Behnken response surface methodology was used to optimize pH (A), inoculum size (B), and fermentation time (C) using a three-factor, three-level approach. The factor levels are shown in Table 1. A total of 17 experiments were conducted, with lactic acid yield as the response value.
[0076] Table 1
[0077]
[0078] Using lactic acid production as the response value, a quadratic prediction model was established through regression analysis, and the resulting three-dimensional response surface analysis is as follows: Figure 15 , Figure 16 , Figure 17 As shown in the figure, the model predicted a lactic acid yield of 3.853 g / L under the conditions of pH=5.64, inoculum size of 11.237%, and fermentation time of 6.546 days. Considering practical feasibility, the conditions were adjusted to pH=5.64, inoculum size of 11%, and fermentation time of 6.5 days for verification. The measured lactic acid yield was 3.816 g / L, with a relative error of only 0.95% compared to the predicted value. The model fit was good, demonstrating high accuracy and reliability.
[0079] In terms of fermentation performance characterization, the Van Soest washing fiber analysis method was used to analyze the fiber composition of the baijiu lees before and after fermentation, and the results are shown in Table 2. After fermentation by KC1 single strain, the cellulose and hemicellulose contents decreased slightly; while after co-culture fermentation with LB9+KC1, the degradation rates of cellulose and hemicellulose reached 56.1% and 68.4%, respectively, and the lignin content also decreased significantly, indicating that the mixed strain system has a synergistic degradation ability for lignocellulose components.
[0080] Table 2
[0081]
[0082] The microstructure of the waste residue before and after fermentation was observed using scanning electron microscopy (SEM). Figure 18 A showed that the surface structure of the lees in the control group before fermentation was dense and smooth, mainly composed of granular materials of relatively uniform size and distribution. After 6.5 days of co-fermentation with LB9+KC1 (…), Figure 18 B Figure 18 C Figure 18 D) Numerous holes, cracks, and erosion marks appeared on the surface of the fermentation trough. A large number of bacterial strains were distributed on the surface, and microbial cells were clearly attached. Filamentous biofilm structures were also visible, indicating vigorous growth and metabolic activity of microorganisms. The microbial community may have improved fermentation efficiency by forming a biofilm to facilitate attachment and growth.
[0083] In summary, this invention, by constructing a KC1 and LB9 co-culture system and combining a staged oxygen supply strategy with response surface methodology, achieves efficient degradation of cellulose and hemicellulose and targeted synthesis of lactic acid using baijiu (Chinese liquor) waste as the sole carbon source, without the need for exogenous enzyme preparations or substrate pretreatment. This method utilizes baijiu industrial waste as raw material, avoids competition with human resources for food, shortens the process flow, reduces production costs, and possesses both resource utilization and environmental friendliness characteristics, demonstrating potential for industrial application.
[0084] The above embodiments are merely preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but which still solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.
Claims
1. A method for preparing lactic acid using a combination of bacterial strains, characterized in that, Includes the following steps: (1) Prepare raw materials and microbial strains. The raw materials are lees from baijiu (Chinese liquor); the microbial strains include Bacillus subtilis. Bacillus subtilis KC1 and Lactobacillus pentosaccharide Lactiplantibacillus pentosus LB9, of which Bacillus subtilis Bacillus subtilis KC1 was deposited on December 11, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 67463), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. (Lactobacillus pentosus) Lactiplantibacillus pentosus LB9 was deposited on November 17, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67311, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. (2) Bacillus subtilis Bacillus subtilis KC1 and Lactobacillus pentosaccharide Lactiplantibacillus pentosus After activation, LB9 strains were mixed to form a composite strain, which was then inoculated into a culture medium using baijiu lees as the sole fermentation substrate without the addition of exogenous enzymes or exogenous excipients. During fermentation, a staged oxygen supply strategy was adopted: aerobic fermentation was carried out for 1–4 days, followed by anaerobic fermentation for 5–9 days. The volume ratio of Bacillus subtilis to Lactobacillus pentosus was 1–3:3–1, and the inoculation amount was 6%–14% of the fermentation medium volume. The amount of baijiu lees added to the fermentation medium was 10 g / L–60 g / L. The initial pH of the fermentation medium was 4.36–6.
36. (3) Collect lactic acid from fermentation products.
2. The method for preparing lactic acid using a combination of bacterial strains according to claim 1, characterized in that, The baijiu lees mentioned are lignocellulose baijiu lees.
3. The method for preparing lactic acid using a combination of bacterial strains according to claim 2, characterized in that, In step (2), aerobic fermentation is carried out for 2 days first, followed by anaerobic fermentation for 5 days.
4. The method for preparing lactic acid using a combination of bacterial strains according to claim 3, characterized in that, In step (2), the Bacillus subtilis Bacillus subtilis KC1 and Lactobacillus pentosaccharide Lactiplantibacillus pentosus The volume ratio of LB9 was 1:1, and the inoculum volume was 11% of the fermentation medium volume.
5. The method for preparing lactic acid using a combination of bacterial strains according to claim 4, characterized in that, In step (2), the initial pH of fermentation is 5.
64.
6. The method for preparing lactic acid using a combination of bacterial strains according to claim 5, characterized in that, In step (2), the amount of baijiu lees added to the fermentation culture medium is 10 g / L.
7. A method for preparing lactic acid using a combination of bacterial strains according to any one of claims 1 to 6, characterized in that, In step (2), before fermentation, Bacillus subtilis is first cultured in LB medium with a pH of 7.
0. Bacillus subtilis KC1 is used for activation; the LB medium consists of: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and distilled water to a final volume of 1000 mL.
8. The method for preparing lactic acid using a combination of bacterial strains according to claim 7, characterized in that, In step (2), before fermentation, Lactobacillus pentosus is first cultured on MRS medium. Lactiplantibacillus pentosus LB9 was activated; the composition of the MRS medium included: 10.0 g peptone, 10.0 g beef extract, 20.0 g glucose, 5.0 g yeast extract, 3.02 g sodium acetate trihydrate, 1.16 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate, 2.0 g triammonium citrate, 0.03 g manganese sulfate monohydrate, and 1.0 mL Tween 80, with distilled water added to bring the volume to 1000 mL.
9. A method for preparing lactic acid using a combination of bacterial strains according to claim 8, characterized in that, The fermentation temperature was 37℃ and the rotation speed was 120 r / min.