Method for co-producing flavone, lignin and L-lactic acid from all components of gordon euryale seed shells

Abstract

The invention belongs to the technical field of resource reutilization, and particularly relates to a method for co-producing flavone, lignin and L-lactic acid from all components of gordon euryale seed shells, which comprises the following steps: mixing the gordon euryale seed shells and a deep eutectic solvent, and carrying out ultrasonic extraction and separation to obtain a first extracting solution and a first residual solid, the first extracting solution containing flavone; the first residual solid and a deep eutectic solvent are mixed, a second extracting solution and a second residual solid are obtained through heating extraction and separation, and the second extracting solution contains lignin; fermenting the second residual solid to obtain L-lactic acid; the eutectic solvent is prepared from the following components: lactic acid, pyrazole and hexadecyl trimethyl ammonium bromide. According to the method disclosed by the invention, full-component utilization of the gordon euryale seed shells is realized, and the extraction rates of flavone, lignin and L-lactic acid are improved.

Description

Technical Field

[0001] This invention belongs to the field of resource recycling technology, specifically relating to a method for the co-production of flavonoids, lignin and L-lactic acid from the whole components of Euryale ferox shell. Background Technology

[0002] Lignocellulose is an extremely abundant biomass resource on Earth, mainly composed of cellulose (30%-50%), hemicellulose (15%-30%), lignin (15%-30%), and extractable components (5%-10%) (Reference: Abolore, RS, S. Jaiswal and AK Jaiswal, Green and sustainable pretreatment methods for cellulose extraction from lignocellulosic biomass and its applications: Areview. Carbohydrate Polymer Technologies and Applications, 2023. 7.). Cellulose is a chain-like polymer rich in β-1,4-glycosidic bonds; hemicellulose is a branched heteropolysaccharide composed of various monosaccharides in different ways; lignin is a polymer formed by phenylpropane structural units linked by C-C and CO bonds; and extractable components are mostly flavonoids, phenols, and alkaloids, which are bioactive components with antibacterial effects and can be widely used in biomedicine, food safety, and other fields. Therefore, how to fully utilize the effective components of lignocellulose is a worthy research topic.

[0003] Euryale ferox, a plant of the Nymphaeaceae family, is widely cultivated in southern China and Southeast Asia. A large amount of the husks remaining after processing are discarded. Euryale ferox husks are a valuable lignocellulose raw material with medicinal and health-promoting properties, making the development and utilization of this resource significant. Currently, research on the development and application of euryale ferox husk raw materials is limited, focusing mainly on the extraction of active ingredients, while a large number of components within the husks are wasted. Therefore, developing technologies that can utilize all components of euryale ferox husks is crucial.

[0004] Currently, most research focuses on the utilization of single components in lignocellulose, leading to the waste of other components. For example, Pinto, E. et al. found that optimizing pretreatment conditions such as temperature and time can only improve the utilization rate of single components such as cellulose or lignin, while other components are discarded (Reference: Pinto, E., et al., Cellulose processing from biomass and its derivatization into carboxymethylcellulose: A review. 2021. 15.). Liu et al. used polyethylene glycol to prepare a eutectic solvent, achieving an 88% cellulose hydrolysis rate and a high lignin extraction rate under sodium salt catalysis (Reference: Liu, L., Q. Li and C. Wan, Deep eutectic solvent-based microextraction system for simultaneous lignocellulose fractionation and furfural production. GreenChemistry. 27(5).); Rodríguez-Jiménez et al. co-utilized hemicellulose and lignin extracted by acid treatment and fractionation (Reference: Rodríguez-Jiménez, S., et al., Valorisation of lignocellulose and low concentration CO2 using a fractionation –photocatalysis – electrolysis process. GREEN CHEMISTRY, 2023. 25(24): p.10611-10621.). It is evident that these studies only utilized cellulose, hemicellulose, and lignin components, while extractable components were not utilized simultaneously.

[0005] To address the issue of fully extracting the effective components, some studies have focused on pretreatment to preferentially separate lignin, followed by the gradual and efficient utilization of cellulose and hemicellulose components. However, this process is complex, requires large quantities of acid and alkali reagents, and is energy-intensive (Reference: Kang, F., et al., New horizons in lignin-first strategies for upgrading lignocellulose. Green Carbon, 2025.). Some studies have prepared various modified catalysts, such as Ni2Al3 alloys and Pt / CeCrO2-x, which can convert cellulose, hemicellulose, and lignin components in biomass into liquid fuels and chemicals, but these require expensive metal materials. Clearly, these methods not only incur high costs but also lead to the loss of extractable components such as hemicellulose, and numerous obstacles remain to the full utilization of lignocellulose components. Therefore, there is an urgent need to explore a green solution that can fully utilize all components of the lignocellulose shell. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the co-production of flavonoids, lignin and L-lactic acid from all components of Euryale ferox shell, thereby realizing the full utilization of Euryale ferox shell and improving the extraction rates of flavonoids, lignin and L-lactic acid.

[0007] This invention provides a method for the co-production of flavonoids, lignin, and L-lactic acid from the whole components of Euryale ferox shell, comprising the following steps: mixing Euryale ferox shell with a eutectic solvent, extracting by ultrasonication, and separating to obtain a first extract and a first residual solid, wherein the first extract contains flavonoids; mixing the first residual solid with a eutectic solvent, extracting by heating, and separating to obtain a second extract and a second residual solid, wherein the second extract contains lignin; and fermenting the second residual solid to obtain L-lactic acid. The eutectic solvent comprises the following components: lactic acid, pyrazole, and hexadecyltrimethylammonium bromide.

[0008] As a preferred embodiment, the molar ratio of lactic acid, pyrazole and hexadecyltrimethylammonium bromide is 1:1:(0.5~20).

[0009] As a preferred embodiment, during ultrasonic extraction and heating extraction, the mass ratio of the eutectic solvent to the gorgon fruit shell is (15~35) g: 1 g.

[0010] As a preferred embodiment, the ultrasonic extraction power is 120~360W; the ultrasonic extraction temperature is 30~70℃; and the ultrasonic extraction time is 60~180min.

[0011] As a preferred embodiment, the temperature for heating and extraction is 80~110℃; the time for heating and extraction is 30~150min.

[0012] As a preferred embodiment, the fermentation includes: stepwise fermentation or simultaneous fermentation; The stepwise fermentation includes the following steps: mixing the second residual solid with cellulase, enzymatically hydrolyzing the supernatant to inactivate the enzyme, adding lactic acid-producing bacteria for fermentation, and obtaining L-lactic acid. The simultaneous fermentation includes the following steps: mixing the second remaining solid with cellulase, enzymatically hydrolyzing it, adding lactic acid-producing bacteria for fermentation, and obtaining L-lactic acid.

[0013] As a preferred embodiment, during stepwise fermentation, the enzymatic hydrolysis temperature is 40~60℃ and the enzymatic hydrolysis time is 60~84h; the fermentation temperature is 38~45℃ and the fermentation time is 42~54h.

[0014] As a preferred embodiment, during simultaneous fermentation, the enzymatic hydrolysis temperature is 40~60℃ and the enzymatic hydrolysis time is 6~18h; the fermentation temperature is 38~45℃ and the fermentation time is 42~54h.

[0015] As a preferred embodiment, the method for preparing the eutectic solvent includes: mixing lactic acid, pyrazole and hexadecyltrimethylammonium bromide, and heating to obtain the eutectic solvent.

[0016] As a preferred embodiment, the heating temperature is 70~90℃ and the heating time is 1~3h.

[0017] Beneficial Effects: This invention provides a method for the co-production of flavonoids, lignin, and L-lactic acid from the whole components of Euryale ferox shells, comprising the following steps: mixing Euryale ferox shells with a eutectic solvent, extracting by ultrasonication, and separating a first extract and a first residual solid, wherein the first extract contains flavonoids; mixing the first residual solid with the eutectic solvent, extracting by heating, and separating a second extract and a second residual solid, wherein the second extract contains lignin; fermenting the second residual solid to obtain L-lactic acid; wherein the eutectic solvent comprises the following components: lactic acid, pyrazole, and hexadecyltrimethylammonium bromide. The eutectic solvent utilized in this invention is a green ionic solution with high selectivity, allowing for the extraction of specific components under different conditions, i.e., selective extraction of flavonoids and lignin, ensuring sufficient extraction of flavonoids and lignin from the material; simultaneously retaining cellulose and hemicellulose components; providing more available sugars; and enabling the production of more L-lactic acid. The results of the examples show that the flavonoid yield can reach a maximum of 17.7% (w / w), the lignin extraction rate is 98.6% (w / w) of the total lignin, and the lactic acid concentration reaches a production concentration of 88.3 g / L with a yield of 99.6% (w / w). The method described in this invention not only achieves the full utilization of the components of the foxnut shell, but also realizes the efficient production of high-value-added products such as flavonoids, lignin, and lactic acid, providing a new technical solution for the graded extraction and utilization of all components of lignocellulose. Attached Figure Description

[0018] Figure 1 To extract the process flow diagram; Figure 2 For glucose standard curve; Figure 3 A standard curve of xylose; Figure 4 This is a standard curve diagram of flavonoids; Figure 5 This is a standard curve for lactic acid. Detailed Implementation

[0019] This invention provides a method for the co-production of flavonoids, lignin, and L-lactic acid from the whole components of Euryale ferox shell, comprising the following steps: mixing Euryale ferox shell with a eutectic solvent, extracting by ultrasonication, and separating to obtain a first extract and a first residual solid, wherein the first extract contains flavonoids; mixing the first residual solid with a eutectic solvent, extracting by heating, and separating to obtain a second extract and a second residual solid, wherein the second extract contains lignin; and fermenting the second residual solid to obtain L-lactic acid. The eutectic solvent comprises the following components: lactic acid, pyrazole, and hexadecyltrimethylammonium bromide.

[0020] Unless otherwise specified, the present invention does not have special requirements for the raw materials used, and commercially available products known to those skilled in the art can be used.

[0021] The eutectic solvent described in this invention refers to a two- or three-component eutectic mixture composed of hydrogen bond acceptors (such as quaternary ammonium salts) and hydrogen bond donors (such as amides, carboxylic acids, and polyols) in a certain stoichiometric ratio, whose freezing point is significantly lower than the melting point of each component pure substance.

[0022] The eutectic solvent of this invention comprises the following components: lactic acid, pyrazole, and hexadecyltrimethylammonium bromide. As a preferred embodiment, the molar ratio of lactic acid, pyrazole, and hexadecyltrimethylammonium bromide can be any value within the range of 1:1:(0.5~20), for example, 1:1:0.5, 1:1:1, 1:1:2, 1:1:5, 1:1:10, or 1:1:20. In the eutectic solvent of this invention, lactic acid and pyrazole have the characteristic of retaining a large amount of cellulose and hemicellulose in the solid during extraction. Simultaneously, the additional addition of hexadecyltrimethylammonium bromide enhances the dissolution of lignin during pretreatment, thereby improving both the lignin extraction rate and the retention rate of cellulose and hemicellulose.

[0023] In a preferred embodiment, the method for preparing the eutectic solvent of the present invention includes: mixing lactic acid (abbreviated as LA), pyrazole (abbreviated as PY), and hexadecyltrimethylammonium bromide (abbreviated as CTAB), and heating to obtain the eutectic solvent; the obtained eutectic solvent is a transparent and clear liquid. The heating temperature of the present invention can be any value within the range of 70~90℃, for example, 70℃, 75℃, 80℃, 85℃, or 90℃; the heating time can be any value within the range of 1~3h, for example, 1h, 1.5h, 2h, 2.5h, or 3h. In a specific embodiment of the present invention, an oil bath is used for heating, as the temperature control of an oil bath is more stable. Heating causes the three solvent groups to dissociate, undergo hydrogen bond recombination, and ultimately form a network. The dissociation of the three solvent groups refers to: CTAB dissociating under the influence of polar molecules LA and PY, releasing free Br⁻; hydrogen bond recombination refers to: the hydrogen bond donor groups (-COOH, -NH-) of LA and PY competitively forming strong hydrogen bonds with Br⁻, while also forming hydrogen bonds with each other; network formation refers to: the formation of a network with Br⁻ as the crosslinking center and LA and PY as connecting units, [CTA]. + It is a disordered and dynamic ternary hydrogen bond network that serves as a filler; that is, heating completes a complex physical self-assembly process dominated by hydrogen bonds.

[0024] As a preferred embodiment, before mixing the water chestnut shells with the eutectic solvent, the present invention pre-treats the water chestnut shells, the pre-treatment including drying and pulverizing. The moisture content of the dried water chestnut shells can be any value within the range of 35% to 45%, for example, 35%, 37%, 40%, 43%, or 45%; the drying temperature can be any value within the range of 70% to 90℃, for example, 70℃, 75℃, 80℃, 85℃, or 90℃; and the drying time can be any value within the range of 8% to 16h, for example, 8h, 10h, 12h, 14h, or 16h. The present invention does not specifically limit the pulverizing method; conventional pulverizing methods are acceptable. In a specific embodiment of the present invention, a hammer mill is used for pulverizing. As a preferred embodiment, the pulverized water chestnut shells are sieved, and the undersize material is used as the pre-treated water chestnut shells. The sieve aperture diameter is any value within the range of 8% to 12mm, for example, 8mm, 10mm, or 12mm.

[0025] This invention involves mixing Euryale ferox shells with a eutectic solvent, followed by ultrasonic extraction to separate a first extract and a first residual solid. The first extract contains flavonoids. During ultrasonic extraction, the mass ratio of the eutectic solvent to the Euryale ferox shells can be any value within the range of (15~35) g:1 g, for example, 15 g:1 g, 20 g:1 g, 25 g:1 g, 30 g:1 g, or 35 g:1 g. Eutectic solvents (DES) can selectively extract flavonoids. Inside cells, multiple interaction sites of DES (hydrogen bond donors, aromatic rings, and cations) simultaneously interact synergistically with multiple functional groups (C=O, aromatic rings, -OH) of the target flavonoid molecule. Hydrogen bond donors directly form hydrogen bonds with C=O, "dissolving" and "stealing" the flavonoid. Pyrazoles containing aromatic rings can form π-π stacking with the aromatic rings of flavonoids face-to-face or face-to-edge, generating additional attraction. CTAB is a surfactant-type component; its long alkyl chains form micro-hydrophobic regions or micelles in DES. The hydrophobic framework of flavonoids can be encapsulated. Encased in these regions, the flavonoids separate from the polar cell wall polysaccharides, enabling recognition and binding. Synergistic effects generate multiple, powerful interactions sufficient to overcome the forces between flavonoid molecules and the plant matrix, "pulling" the flavonoids into the eutectic solvent to form a stable "eutectic solvent-flavonoid" complex, achieving dissolution and separation. Because other major plant components (such as starch and cellulose) lack the strong hydrogen bond acceptors (C=O) and large aromatic hydrophobic frameworks of flavonoids, their interactions with the eutectic solvent are weaker, and therefore most remain in the residue, achieving highly selective extraction of flavonoids. Simultaneously, the remaining solid residue retains lignin, cellulose, and hemicellulose components to the greatest extent possible. The ultrasonic extraction power described in this invention can be any value within the range of 120~360W, for example, 120W, 180W, 240W, 300W, or 360W; the ultrasonic extraction temperature can be any value within the range of 30~70℃, for example, 30℃, 40℃, 50℃, 60℃, or 70℃; the ultrasonic extraction time can be any value within the range of 60~180min, for example, 60min, 90min, 120min, 150min, or 180min. Under ultrasonic conditions, the dissolution of active ingredients such as flavonoids in the material can be promoted, and a good extraction effect of flavonoids can be achieved even at relatively low temperatures. This invention does not have a special limitation on the separation method; conventional separation methods can be used. In a specific embodiment of this invention, vacuum filtration is performed using a Buchner funnel.

[0026] This invention involves mixing a first residual solid with a eutectic solvent, followed by heating for extraction, resulting in a second extract and a second residual solid. The second extract contains lignin. During heating extraction, the eutectic solvent selectively extracts lignin at higher temperatures. At high temperatures, lignin is released from the tightly bound structure of the biomass. Lignin also contains strong hydrogen bond acceptors (C=O) and a large aromatic hydrophobic framework, which interact strongly with the eutectic solvent, thus allowing for selective extraction. Simultaneously, the second residual solid retains cellulose and hemicellulose components, providing more sugar for subsequent fermentation. In this invention, the mass ratio of the eutectic solvent to the gorgon fruit shell during heating extraction can be any value within the range of (15~35) g:1 g, for example, 15 g:1 g, 20 g:1 g, 25 g:1 g, 30 g:1 g, or 35 g:1 g. The heating extraction temperature described in this invention can be any value within the range of 80~110℃, for example, 80℃, 90℃, 100℃, or 110℃; the heating extraction time can be any value within the range of 30~150min, for example, 30min, 60min, 90min, 120min, or 150min. In a specific embodiment of this invention, an oil bath is used for heating. After the two-step extraction process, flavonoids and lignin in the material are fully extracted, while cellulose and hemicellulose are largely retained in the second residual solid. On the one hand, the cellulose and hemicellulose content in the residual solid increases significantly; on the other hand, because lignin is separated from the second residual solid, the resistance to the reaction between cellulase and cellulose is reduced, which is beneficial to the enzymatic hydrolysis step in the subsequent fermentation process.

[0027] This invention yields L-lactic acid through fermentation of a second residual solid. In a preferred embodiment, the fermentation includes either stepwise or simultaneous fermentation. In a preferred embodiment, the stepwise fermentation includes the following steps: mixing the second residual solid with cellulase, enzymatically hydrolyzing the mixture, retaining the supernatant to inactivate the enzyme, adding lactic acid-producing bacteria for fermentation, and obtaining L-lactic acid. The volume-to-mass ratio of cellulase to the second residual solid can be any value within the range of (0.04~0.05) mL:1g, for example, 0.04mL:1g, 0.046mL:1g, or 0.05mL:1g. The cellulase activity can be any value within the range of 220~270 FPU / mL, for example, 220, 250, or 270 FPU / mL. In a specific embodiment of this invention, the cellulase activity is 256.0 FPU / mL. In this invention, during stepwise fermentation, the enzymatic hydrolysis temperature can be any value within the range of 40-60℃, for example, 40℃, 50℃, or 60℃; the enzymatic hydrolysis time can be any value within the range of 60-84h, for example, 60h, 72h, or 84h. In a specific embodiment of this invention, the results of stepwise saccharification and fermentation show that the concentrations of glucose and xylose obtained after enzymatic hydrolysis of the material after two-step low-cosolvent treatment are significantly higher than those of the material after only one-step low-cosolvent treatment. After enzymatic hydrolysis, the supernatant is retained. After enzyme inactivation, lactic acid-producing bacteria are added to the supernatant for fermentation. The amount of lactic acid-producing bacteria added in this invention is any value within the range of 8%-12% of the second remaining solid volume, for example, 8%, 10%, or 12%. The lactic acid-producing bacteria in this invention may include: Bacillus coagulans or Pediococcus lactis. The fermentation temperature can be any value within the range of 38-45℃, for example, 38℃, 42℃, or 45℃; the fermentation time can be any value within the range of 42-54h, for example, 42h, 48h, or 54h. Stepwise fermentation involves first enzymatically hydrolyzing the cellulose and hemicellulose in the material to convert them entirely into the corresponding monosaccharides, then inactivating the enzymes before fermentation with lactic acid-producing bacteria. As another preferred embodiment, the simultaneous fermentation includes the following steps: mixing the second residual solid with cellulase, enzymatically hydrolyzing it, and then adding lactic acid-producing bacteria for fermentation to obtain L-lactic acid. The mass ratio of cellulase to the second residual solid in this invention can be any value within the range of (0.04~0.05) mL:1g, for example, 0.04mL:1g, 0.046mL:1g, or 0.05mL:1g. The cellulase activity in this invention can be any value within the range of 220~270 FPU / mL, for example, 220, 250, or 270 FPU / mL. In a specific embodiment of this invention, the cellulase activity is 256.0 FPU / mL. In the simultaneous fermentation process of this invention, the enzymatic hydrolysis temperature is any value within the range of 40~60℃, for example, 40℃, 50℃ or 60℃; the enzymatic hydrolysis time can be any value within the range of 6~18h, for example, 6h, 12h or 18h.The amount of lactic acid-producing bacteria added in this invention is any value within the range of 8% to 12% of the volume of the second remaining solid, for example, 8%, 10%, or 12%. The lactic acid-producing bacteria in this invention may include: *Bacillus coagulans* or *Pediococcus lactis*. The fermentation temperature is any value within the range of 38 to 45°C, for example, 38°C, 42°C, or 45°C; the fermentation time can be any value within the range of 42 to 54 hours, for example, 42 hours, 48 ​​hours, or 54 hours. Simultaneous fermentation means that after brief saccharification, without enzyme inactivation treatment, lactic acid-producing bacteria are added, and fermentation proceeds simultaneously with the enzymatic hydrolysis process. After extraction using a eutectic solvent in the above two steps, a large amount of cellulose and hemicellulose components are retained in the second remaining solid, allowing the strain to utilize more sugars during fermentation and produce more L-lactic acid.

[0028] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a method for the co-production of flavonoids, lignin, and L-lactic acid from the whole components of Euryale ferox shell provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1 1. Raw material pretreatment The shells of the water chestnut were collected from Jiangsu Province, China, and dried in an oven at 80℃ for 12 hours. After drying, the moisture content of the shells was 40.1%. Following drying, the shells were pulverized using a hammer mill and passed through a sieve with an aperture of approximately 10 mm to obtain powdered water chestnut shells for subsequent experiments. (All raw materials used in the following examples were obtained from the same batch of pre-processed materials.) 2. Preparation of eutectic solvent (abbreviated as [LA][PY][CTAB]) Preparation of [LA][PY][CTAB]: Pyrazole (PY), lactic acid (LA), and hexadecyltrimethylammonium bromide (CTAB) were mixed in a 250 mL Erlenmeyer flask at a molar ratio of 1:1:0.5. The mixture was then placed in an oil bath and heated at 80 °C for 2 h with stirring. Once a clear and transparent liquid was formed, [LA][PY][CTAB] was obtained. The mixture was then cooled to room temperature for later use.

[0030] 3. Extraction of flavonoids using [LA][PY][CTAB]-assisted ultrasonic treatment. Take 1g of the Euryale ferox shell powder obtained in step 1 and mix it with 15g of [LA][PY][CTAB] obtained in step 2 in an Erlenmeyer flask (the liquid-to-solid ratio of [LA][PY][CTAB] to Euryale ferox shell powder is 15g:1g). Place the Erlenmeyer flask in an ultrasonic extractor for ultrasonic extraction at a power of 120W, a time of 60min, and a temperature of 70℃. After extraction, filter the mixture through a Buchner funnel to obtain an extract containing flavonoids.

[0031] 4. Lignin extraction using [LA][PY][CTAB]-assisted high-temperature treatment. The remaining solid residue from step 3 was mixed with 15g of the [LA][PY][CTAB] obtained in step 2 in a test tube (the liquid-to-solid ratio of [LA][PY][CTAB] to Euryale ferox shells was 15g:1g). The mixture was placed in an oil bath for reaction at 70℃ for 30 minutes. After the reaction, the mixture was filtered through a Buchner funnel to obtain an extract containing lignin; the remaining solid residue was used for subsequent enzymatic hydrolysis and lactic acid fermentation.

[0032] 5. Activation culture of bacterial strains Bacillus coagulans CGMCC 1.3220 was used as the L-lactic acid producing bacterium. Five single colonies were picked and inoculated into seed culture medium, and cultured at 42°C and 150 rpm for 24 h to obtain the bacterial suspension. The bacterial suspension was then inoculated into MRS medium at an inoculum volume of 10% (v / v) and fermented at 42°C and 150 rpm for 48 h to obtain activated Bacillus coagulans (OD600=1.0). During the culture process, the pH was maintained at 5.5 using calcium carbonate.

[0033] Seed culture medium components: 20 g / L glucose, 2.5 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen phosphate, 10 g / L peptone, 10 g / L yeast extract, 0.25 g / L manganese sulfate monohydrate, 6 g / L calcium carbonate, 0.2925 g / L magnesium sulfate heptahydrate, and 1.0075 g / L diammonium hydrogen citrate.

[0034] MRS culture medium components: 20 g / L glucose, 5 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen citrate, 0.58 g / L magnesium sulfate heptahydrate, 2 g / L dipotassium hydrogen phosphate, 10 g / L peptone, 10 g / L yeast extract and 0.25 g / L manganese sulfate monohydrate.

[0035] 6. Stepwise fermentation preparation of L-lactic acid bacteria Mix 0.5g of the remaining solid filter residue from step 4, 0.023mL of cellulase Cellic CTec2 (product: total protein concentration 86.3mg proteins / mL, cellulase activity 256.0FPU / mL), 10mL of citrate buffer (0.1mol / L, pH 4.8), and 80µL of tetracycline solution (concentration 10mg / mL) in a 100mL Erlenmeyer flask. Incubate at 50℃ and 150rpm for 72h in a constant temperature shaker. After sterilizing the supernatant, obtain the hydrolysate for later use. Prepare the MRS medium according to the composition, using the hydrolysate instead of glucose, while keeping other components unchanged. Inoculate with 10% (v / v) of the activated Bacillus coagulans from step 5 and ferment at 42℃ for 48h in a constant temperature shaker at 200rpm to obtain L-lactic acid. During fermentation, the pH was maintained at 5.5 using calcium carbonate.

[0036] Example 2 The procedure was carried out as in Example 1, except that in step 2, the molar ratio of pyrazole, lactic acid and hexadecyltrimethylammonium bromide was replaced with 1:1:1.

[0037] Example 3 The procedure was carried out as in Example 1, except that in step 2, the molar ratio of pyrazole, lactic acid and hexadecyltrimethylammonium bromide was replaced with 1:1:2.

[0038] Example 4 The procedure was carried out as in Example 1, except that in step 2, the molar ratio of pyrazole, lactic acid and hexadecyltrimethylammonium bromide was replaced with 1:1:5.

[0039] Example 5 The procedure was carried out as in Example 1, except that in step 2, the molar ratio of pyrazole, lactic acid and hexadecyltrimethylammonium bromide was replaced with 1:1:20.

[0040] Example 6 The procedure was carried out in accordance with Example 2, except that in step 3, the liquid-to-material ratio of [LA][PY][CTAB] to Euryale ferox shell powder was replaced with 20g:1g.

[0041] Example 7 The procedure was carried out in accordance with Example 2, except that in step 3, the liquid-to-material ratio of [LA][PY][CTAB] to Euryale ferox shell powder was replaced with 25g:1g.

[0042] Example 8 The procedure was carried out in accordance with Example 2, except that in step 3, the liquid-to-material ratio of [LA][PY][CTAB] to Euryale ferox shell powder was replaced with 30g:1g.

[0043] Example 9 The procedure was carried out in accordance with Example 2, except that in step 3, the liquid-to-material ratio of [LA][PY][CTAB] to Euryale ferox shell powder was replaced with 35g:1g.

[0044] Example 10 The procedure is carried out in accordance with Example 7, except that in step 3, the ultrasonic power is replaced with 180W.

[0045] Example 11 The procedure is carried out in accordance with Example 7, except that in step 3, the ultrasonic power is replaced with 240W.

[0046] Example 12 The procedure is carried out in accordance with Example 7, except that in step 3, the ultrasonic power is replaced with 300W.

[0047] Example 13 The procedure is carried out in accordance with Example 7, except that in step 3, the ultrasonic power is replaced with 360W.

[0048] Example 14 The procedure is carried out in accordance with Example 10, except that in step 3, the ultrasound time is replaced with 90 minutes.

[0049] Example 15 The procedure is carried out in accordance with Example 10, except that in step 3, the ultrasound time is replaced with 120 min.

[0050] Example 16 The procedure is carried out in accordance with Example 10, except that in step 3, the ultrasound time is replaced with 150 min.

[0051] Example 17 The procedure is carried out in accordance with Example 10, except that in step 3, the ultrasound time is replaced with 180 min.

[0052] Example 18 The procedure is carried out in accordance with Example 15, except that in step 3, the ultrasonic temperature is replaced with 60°C.

[0053] Example 19 The procedure is carried out in accordance with Example 15, except that in step 3, the ultrasonic temperature is replaced with 50°C.

[0054] Example 20 The procedure is carried out in accordance with Example 15, except that in step 3, the ultrasonic temperature is replaced with 40°C.

[0055] Example 21 The procedure is carried out in accordance with Example 15, except that in step 3, the ultrasonic temperature is replaced with 30°C.

[0056] Experimental Example 1 1. Determination of cellulose, hemicellulose and lignin The contents of cellulose and hemicellulose were determined using a two-step acid hydrolysis method. After the two-step acid hydrolysis, glucose and xylose in the hydrolysate were analyzed by HPLC. Cellulose content (%) = (glucose concentration × reaction liquid volume × 1.111) / weight of water chestnut shell × 100; hemicellulose content (%) = (xylose concentration × reaction liquid volume × 1.136) / weight of water chestnut shell × 100. (Reference: Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008. Determination of sugars, byproducts, and degradation products in liquid fraction process samples. National Renewable Energy Laboratory Technical Report NREL / TP-510-42623. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2012. Determination of structural carbohydrates and lignin in biomass. National Renewable Energy Laboratory Technical Report NREL / TP-510-42618. The standard curves for glucose and xylose are shown below. Figure 2 and 3 .

[0057] The cellulose content of the gorgon fruit shell powder in step 1 of the example was determined to be 27.2% (w / w), the hemicellulose content was 14.6% (w / w), and the lignin content was 35.2% (w / w), which were used in subsequent calculation formulas to correspond to the content of each component in untreated gorgon fruit shells.

[0058] When determining the content of components at each stage after extraction, the content of cellulose, hemicellulose and lignin in the sample is determined by a two-step acid hydrolysis method, and the retention rate is calculated according to the following formula.

[0059] Cellulose retention rate (%) = (mass of treated gorgon fruit shells × cellulose content of treated gorgon fruit shells) / (mass of untreated gorgon fruit shells × cellulose content of untreated gorgon fruit shells) × 100.

[0060] Hemicellulose retention rate (%) = (mass of treated gorgon fruit shells × hemicellulose content of treated gorgon fruit shells) / (mass of untreated gorgon fruit shells × hemicellulose content of untreated gorgon fruit shells) × 100.

[0061] Lignin retention rate (%) = (mass of treated water chestnut shells × lignin content of treated water chestnut shells) / (mass of untreated water chestnut shells × lignin content of untreated water chestnut shells) × 100.

[0062] 2. Determination of flavonoid concentration and antioxidant activity Prepare a 0.20 mg / mL standard solution by adding 0.01 g of rutin to 50 mL of 50% ethanol (v / v). In six 25 mL volumetric flasks, first add 1 mL of 5% sodium nitrite solution (w / w), then add 0.5, 1.0, 2.0, 4.0, and 8.0 mL of the standard solution respectively, shake well, and let stand for 6 min. Next, add 1 mL of 10% aluminum nitrate solution (w / w) to each flask, mix well, and let stand for 6 min. Finally, add 10 mL of 4% sodium hydroxide solution (w / w) to each flask, then dilute to volume with 50% ethanol and incubate in the dark for 15 min. Measure the absorbance at 510 nm and plot a standard curve of absorbance versus concentration (e.g., [missing information]). Figure 4 The standard curve formula is y=0.5736x. The absorbance of the extract was measured using a UV spectrophotometer, and the flavonoid content in the extract was calculated based on the standard curve. Three replicate experiments were performed, and the flavonoid yield was calculated using the following formula.

[0063] Flavonoid yield (%) = Flavonoid content extracted (g) / Flavonoid content of unextracted Euryale ferox shell (g) × 100.

[0064] I. First, under the same initial conditions (liquid-to-solid ratio 15:1, ultrasonic power 120W, ultrasonic time 60min, ultrasonic temperature 70℃), the effects of different [LA][Py][CTAB] molar ratios (1:1:0.5, 1:1:1, 1:1:2, 1:1:5 or 1:1:20) on the yield of flavonoids and the retention rates of cellulose, hemicellulose and lignin were investigated. The detection methods described in 2 and 3 above were used to detect the results of Examples 1 to 5. The results are shown in Table 1.

[0065] Table 1 Effect of different molar ratios on yield

[0066] Note: Other conditions are: liquid-to-material ratio 15:1, ultrasonic power 120W, ultrasonic time 60min, and ultrasonic temperature 70℃.

[0067] As shown in Table 1, the flavonoid yield first increased and then decreased with the increase of CTAB content. The highest flavonoid yield was observed at a molar ratio of 1:1:1, along with relatively high retention rates of lignin, cellulose, and hemicellulose. Therefore, a molar ratio of 1:1:1 was selected as the subsequent reaction condition.

[0068] II. Next, the effect of different liquid-to-solid ratios (w / w) (15:1, 20:1, 25:1, 30:1 or 35:1) was investigated. That is, the detection methods in 2 and 3 above were used to test Examples 2 and 6-9. The results are shown in Table 2.

[0069] Table 2 Effect of different feed-to-liquid ratios on yield

[0070] Note: Other conditions are: molar ratio 1:1:1, ultrasonic power 120W, ultrasonic time 60min, ultrasonic temperature 70℃.

[0071] As can be seen from Table 2, the liquid-to-solid ratio has a certain impact on the retention rates of cellulose, hemicellulose and lignin. When the liquid-to-solid ratio is 25g:1g, the retention rates of the three are the highest. Therefore, a liquid-to-solid ratio of 25g:1g is selected as the subsequent reaction condition.

[0072] III. Finally, the effects of different powers (120W, 180W, 240W, 300W or 360W), time (60min, 90min, 120min, 150min or 180min), and temperature (30℃, 40℃, 50℃, 60℃ or 70℃) during ultrasonic treatment were gradually investigated. That is, the detection methods in 2 and 3 above were used to test Examples 7 and Examples 10 to 21, and the results are shown in Tables 3 to 5.

[0073] Table 3 Effect of different ultrasound powers on flavonoid yield

[0074] Note: Other conditions are: molar ratio 1:1:1, liquid-to-solid ratio 25:1, ultrasonic time 60 min, ultrasonic temperature 70℃.

[0075] Table 4. Effect of different ultrasound times on flavonoid yield

[0076] Note: Other conditions are: molar ratio 1:1:1, liquid-to-solid ratio 25:1, ultrasonic power 180W, ultrasonic temperature 70℃.

[0077] Table 5. Effect of different ultrasonic temperatures on flavonoid yield

[0078] Note: Other conditions are: molar ratio 1:1:1, liquid-to-solid ratio 25:1, ultrasonic power 180W, and ultrasonic time 120min.

[0079] As can be seen from Tables 3-5, when the ultrasonic temperature is 70℃, the power is 180W, and the time is 120min, the yield of flavonoids can reach the highest level of 17.7% (w / w). At the same time, the cellulose, hemicellulose and lignin components are also retained to the greatest extent, with retention rates of 99.6% (w / w), 99.7% (w / w) and 99.5% (w / w), respectively.

[0080] Experimental Example 2 12 mg of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH•) was placed in a 100 mL volumetric flask, dissolved in 95% ethanol, and diluted to volume to prepare a DPPH• solution for determining the DPPH• scavenging rate (antioxidant activity) of the flavonoid solution. A 0.384 g / L ABTS•⁺ solution and a 1.35 g / L potassium persulfate solution were mixed, allowed to stand in the dark for 12 h, and then diluted 16 times to determine the ABTS•⁺ scavenging rate (antioxidant activity) of the flavonoid solution. The •OH scavenging rate (antioxidant activity) of the flavonoid solution was determined using a 6 mmol / L H₂O₂ solution, a 3 mmol / L pyrogallol solution, and a 6 mmol / L salicylic acid solution. A vitamin C standard solution (CAS No. 50-81-7) was used as the control group instead of the flavonoid solution. The DPPH•, ABTS•⁺, and •OH scavenging rates (antioxidant activity) of the flavonoid extract and the vitamin C standard solution at corresponding concentrations were compared. Methods for determining DPPH scavenging rate (antioxidant activity) (Reference: Farshchi-Andisi, A., Salami, M., Miran, M., Siloto, NA, Askari, G., Emam-Djomeh, Z., & Saldana, MDA, 2025. Hydrogel composite for curcuminencapsulation using whey protein isolate and arabinoxylan extracted from sesame hull waste. Int. J. Biol. Macromol. 145575.); Methods for determining ABTS scavenging rate (antioxidant activity) (Reference: Munteanu, IG, Apetrei, C., 2021. Analytical methods used in determining antioxidant activity: A Review. Int. J. Mol. Sci. 22 (7),3380.); Methods for determining OH scavenging rate (antioxidant activity) (Reference: Gulcin, I., 2025. Antioxidants: a comprehensive review.) Arch. Toxicol. 99 (5), 1893-1997.).

[0081] The antioxidant activity of the flavonoid extract in Example 15 was comprehensively evaluated by its scavenging rate of DPPH•, ABTS•⁺, and •OH (hydroxyl radicals). The results are shown in Tables 6-8.

[0082] Table 6. DPPH• scavenging rate of extracted flavonoids

[0083] Table 7 •OH scavenging rate of extracted flavonoids

[0084] Table 8. ABTS•⁺ scavenging rate of extracted flavonoids

[0085] As shown in Tables 6-8, the flavonoid-containing extract of Example 15 exhibits stronger DPPH•, ABTS•⁺, and •OH (hydroxyl radical) scavenging abilities compared to the standard vitamin C solution. In conclusion, the flavonoid solution extracted from *Euryale ferox* shells via [LA][PY][CTAB]-assisted ultrasonic treatment possesses excellent and comprehensive antioxidant activity.

[0086] Example 22 The procedure was carried out in accordance with Example 15, except that in step 4, the reaction temperature was replaced with 80°C.

[0087] Example 23 The procedure was carried out in accordance with Example 15, except that in step 4, the reaction temperature was replaced with 90°C.

[0088] Example 24 The procedure was carried out in accordance with Example 15, except that in step 4, the reaction temperature was replaced with 100°C.

[0089] Example 25 The procedure was carried out in accordance with Example 15, except that in step 4, the reaction temperature was replaced with 110°C.

[0090] Example 26 The reaction was carried out in accordance with Example 25, except that in step 4, the reaction temperature was replaced with 60 min.

[0091] Example 27 The procedure was carried out in accordance with Example 25, except that in step 4, the reaction temperature was replaced with 90 min.

[0092] Example 28 The procedure was carried out in accordance with Example 25, except that in step 4, the reaction temperature was replaced with 120 min.

[0093] Example 29 The reaction was carried out in accordance with Example 25, except that in step 4, the reaction temperature was replaced with 150 min.

[0094] Experimental Example 3 The lignin-containing extract obtained in step 4 of Example 1 was mixed with acidified water (pH=2) at a ratio of 3:1 (v / v), magnetically stirred for 15 minutes, and then allowed to stand overnight at 4°C for approximately 12 hours. The precipitated lignin was obtained by filtration, washed three times with distilled water, and dried in an oven at 50°C for 24 hours. The moisture content after drying was 10.2% (w / w). The lignin content was determined by a two-step acid hydrolysis method, and the lignin extraction rate was calculated using the following formula.

[0095] Lignin extraction rate (%) = Extracted lignin content / (Lignin content of unextracted gorgon fruit shells × Lignin content of extracted gorgon fruit shells) × 100%.

[0096] Using the method of Example 1, the retention rates of cellulose and hemicellulose in the lignin-containing extract obtained in step 4 of Example 1 were tested, and the results are shown in Tables 9 and 10.

[0097] Table 9. Effect of different temperatures on lignin extraction rate

[0098] Note: Other conditions are: molar ratio 1:1:1, liquid-to-solid ratio 15:1, heating time 30 min.

[0099] Table 10 Effect of different time points on lignin extraction rate

[0100] Note: Other conditions are: molar ratio 1:1:1, liquid-to-solid ratio 15:1, and heating temperature 110℃.

[0101] As shown in Table 9, the lignin extraction rate increases with increasing temperature. The lignin extraction rate reaches its maximum at 110℃, while the retention rates of cellulose and hemicellulose are also relatively high. Therefore, 110℃ is chosen as the subsequent reaction condition.

[0102] As shown in Table 10, the extraction rate of lignin first increases and then decreases with the extension of reaction time. At 90 min, the extraction rate of lignin reaches the highest level of 98.6% (w / w), while the retention rate of cellulose is 98.1% and the retention rate of hemicellulose is 98.2% (w / w).

[0103] Therefore, the optimal reaction conditions are a heating temperature of 110℃ and a reaction time of 120 min in step 4.

[0104] Example 29 The process was carried out in accordance with Example 28, except that step 6 was a simultaneous fermentation, in which 0.5 g of solid residue and 0.023 mL of cellulase Cellic CTec2 (total protein concentration 86.3 mg proteins / mL, enzyme activity 256.0 FPU / mL) were added to MRS medium without added glucose, and saccharification was carried out at 50 °C and 150 rpm for 12 h. Then, 10% (v / v) of Bacillus coagulans activated in step 5 was inoculated, and fermentation was carried out at 42 °C for 48 h and cultured in a constant temperature shaker at 200 rpm to obtain L-lactic acid.

[0105] Test Example 4 L-lactic acid was detected by high-performance liquid chromatography (HPLC), following the method described in the article: The mobile phase was 5 mM H₂SO₄ solution, delivered at a flow rate of 0.6 mL / min, and the column temperature was maintained at 65 ℃. (Qiu et al., 2023). The curve for obtaining L-lactic acid is shown in the figure. Figure 5 Meanwhile, the content of cellulose and hemicellulose in the shell of the water chestnut was determined using the method of Experiment 1, and the yield of L-lactic acid was calculated according to the following formula.

[0106] L-lactic acid yield (%) = (volume of fermentation broth × L-lactic acid concentration) / (mass of foxnut shell × cellulose content × 1.111 + mass of foxnut shell × hemicellulose content × 1.136) × 100.

[0107] The solid residue of water chestnut shells after two-step [LA][PY][CTAB] treatment still retains a large amount of cellulose and hemicellulose, making it essential to fully utilize them. Untreated water chestnut shells were used as a control. Simultaneously, the fermentation of L-lactic acid in the remaining solid residues of steps 3 and 4 was tested using the stepwise fermentation method of Example 28. The simultaneous fermentation method of Example 29 was also used to test the L-lactic acid fermentation of the remaining solid residues of steps 3 and 4. The results are shown in Table 11.

[0108] Table 11 Effects of different fermentation methods on L-lactic acid yield

[0109] Table 11 shows that the results of stepwise saccharification and fermentation indicate that the concentrations of glucose and xylose obtained after enzymatic hydrolysis of the material treated with two steps of [LA][PY][CTAB] were significantly higher than those obtained after only the first step of [LA][PY][CTAB] treatment. The final lactic acid concentration obtained from the fermentation of the material treated with two steps of [LA][PY][CTAB] treatment (75.5 g / L) was also significantly higher than that obtained after only the first step of [LA][PY][CTAB] treatment (48.9 g / L). The results of simultaneous saccharification and fermentation indicate that the final lactic acid concentration obtained from the fermentation of the material treated with two steps of [LA][PY][CTAB] treatment was 88.3 g / L, with a yield of 99.6%, significantly higher than the fermentation level of the material treated with only the first step of [LA][PY][CTAB] treatment.

[0110] Therefore, the method of this invention, using ultrasonic treatment assisted by a eutectic solvent, can completely extract flavonoids from the shell of *Euryale ferox*, achieving an extraction yield of 17.7% (w / w) of the dry weight of the material. Further high-temperature treatment assisted by a eutectic solvent efficiently extracts the lignin component from the remaining solid, achieving an extraction rate of 98.6% (w / w) of the total lignin. Finally, through simultaneous saccharification and fermentation, 98.1% (w / w) of the cellulose and 98.2% (w / w) of the hemicellulose in the solid are converted into lactic acid, reaching a lactic acid concentration of 88.3 g / L, with a yield of 99.6% (w / w). This invention has significant implications for the full utilization of other biomass components.

[0111] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for co-production of flavonoids, lignin and L-lactic acid from whole Euryale ferox shell, characterized in that, The method comprises the following steps: mixing the Euryale shell and a deep eutectic solvent, and separating a first extract liquid and a first residual solid by ultrasonic extraction, wherein the first extract liquid contains flavones; mixing the first residual solid and the deep eutectic solvent, and separating a second extract liquid and a second residual solid by heating extraction, wherein the second extract liquid contains lignin; and obtaining L-lactic acid by fermenting the second residual solid. The deep eutectic solvent comprises the following components: lactic acid, pyrazole and cetyltrimethylammonium bromide.

2. The method of claim 1, wherein, The molar ratio of the lactic acid, the pyrazole and the cetyltrimethylammonium bromide is 1:1:(0.5-20).

3. The method according to claim 1 or 2, characterized in that, The mass ratio of the deep eutectic solvent to the Euryale shell is (15-35) g:1 g during the ultrasonic extraction and the heating extraction.

4. The method of claim 1, wherein, The ultrasonic extraction is performed at a power of 120-360 W, a temperature of 30-70 ℃ and for 60-180 min.

5. The method of claim 1, wherein, The heating extraction is performed at a temperature of 80-110 ℃ and for 30-150 min.

6. The method of claim 1, wherein, The fermentation comprises step-by-step fermentation or synchronous fermentation. The step-by-step fermentation comprises the following steps: mixing the second residual solid and cellulase, retaining the supernatant after enzymolysis, adding lactic acid-producing bacteria to ferment, and obtaining L-lactic acid. The synchronous fermentation comprises the following steps: mixing the second residual solid and cellulase, adding lactic acid-producing bacteria to ferment after enzymolysis, and obtaining L-lactic acid.

7. The method of claim 6, wherein, During the step-by-step fermentation, the enzymolysis is performed at a temperature of 40-60 ℃ and for 60-84 h, and the fermentation is performed at a temperature of 38-45 ℃ and for 42-54 h.

8. The method of claim 6, wherein, During the synchronous fermentation, the enzymolysis is performed at a temperature of 40-60 ℃ and for 6-18 h, and the fermentation is performed at a temperature of 38-45 ℃ and for 42-54 h.

9. The method of claim 1 or 2, wherein, The preparation method of the deep eutectic solvent comprises the following steps: mixing lactic acid, pyrazole and cetyltrimethylammonium bromide, and obtaining the deep eutectic solvent after heating.

10. The method of claim 9, wherein, The heating is performed at a temperature of 70-90 ℃ and for 1-3 h.

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