Preparation method of flavanone polyphenol nutritional wine with pine fragrance

Abstract

The invention discloses a preparation method of flavanone polyphenol nutritional wine with pine fragrance. The larch tree sawdust is used as a raw material source of flavanone polyphenol, and is a breathable and loose auxiliary material in wine brewing. The method comprises the following steps: mixing pine sawdust with wine brewing grains, steaming and moistening, adding distiller's yeast and combined enzyme, and gradually dissolving dihydroquercetin and other dihydroflavone polyphenol substances separated out from cellulose of the pine sawdust subjected to enzymolysis by the combined enzyme into fermented grains along with fermentation by adopting a pure grain white wine solid-state wine brewing process in the brewing fermentation process; introducing saturated steam to promote the transfer of the extract; and carrying out the procedures of distilling to take wine, separating vinasse, regulating acid, crystallizing, redissolving and the like to prepare the flavanone polyphenol nutritional wine with pine fragrance. Flavonoid polyphenols generated in the wine brewing process are redissolved in the wine, so that the wine is rich in dihydroquercetin, soft in taste, easy to accept and unique in pine aroma, pollution of reagents such as industrial ethanol and methanol is avoided in the whole preparation process, and the food safety is high.

Description

Technical Field

[0001] This invention belongs to the field of nutritional wine production technology. Based on the pure grain brewing process for baijiu (Chinese white liquor), this invention specifically describes a method for preparing a pine-flavored dihydroflavonoid polyphenol nutritional wine. Background Technology

[0002] Dihydroquercetin (C 15 H 12 O7 (dihydroquercetin) is an important flavonoid compound found in nature, belonging to the dihydroflavonoid polyphenol compound family. It is readily soluble in ethanol and acetic acid, soluble in boiling water, slightly soluble in cold water, and insoluble in benzene. It possesses good antioxidant and antitumor biological activities and is widely used in health care and medical fields. It is mainly used to treat alcoholic fatty liver disease, coronary artery disease, and for antitumor purposes. Studies have shown that the antioxidant capacity of dihydroquercetin is approximately 500 times that of vitamin C, 350 times that of vitamin E, and 95 times that of coenzyme Q10. Flavonoid polyphenol compounds have been extracted from plants such as yew, paulownia, and larch, with dihydroquercetin, also known as taxol, being the main component. Currently, it has been confirmed that dihydroflavonoid polyphenol compounds are most abundant in larch roots. Existing research indicates that the dihydroflavonoid polyphenol compounds extracted from Siberian larch (Larix sibirica) include: dihydroquercetin (taxanthin) 92.36%, linalool (dihydrokaempferol) 2.99%, hesperidin 0.198%, quercetin 0.436%, naringenin 0.26%, kaempferol 0.06%, pinophytic acid 0.088%, totaling 96.392%.

[0004] Dihydroquercetin is currently obtained primarily through three pathways: chemical synthesis, biosynthesis, and extraction. Currently, extraction methods are used both domestically and internationally to extract dihydroquercetin from natural plants, including enzyme-induced extraction, ethanol solution dissolution, ultrasonic-microwave extraction, macroporous resin enrichment and purification, silica gel column chromatography, and polyamide adsorption extraction. However, most existing dihydroquercetin extraction methods leave excessive organic solvent residues, and even residues of carcinogenic solvents and high molecular weight compounds.

[0005] For example, Chinese patent application number CN201310302501.0 uses 95% ethanol and methyl tert-butyl ether to extract dihydroquercetin from Larix gmelinii. Methyl tert-butyl ether is listed as a Group 3 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization. Furthermore, methyl tert-butyl ether is highly soluble in water, making it difficult to remove when combined with aqueous ethanol.

[0006] Other literature reports that methanol reflux extraction followed by dextran gel column separation yielded total flavonoid extracts from larch, including dihydroquercetin and sennaol. Methanol is also a toxic solvent; when ingested, it is converted into formaldehyde and formic acid, posing health risks, potentially leading to blindness, organ failure, or even death.

[0007] Chinese patents with application numbers CN 201710359876, CN201811502285, and CN202210409455 employ extraction with ethanol of 95% or higher concentration, followed by purification using high-molecular-weight polyamide polymers or macroporous resins. Solvents such as n-hexane and methyl tert-butyl ether are used to remove pine resin. The 95% or higher concentration of industrial ethanol used in the extraction process typically contains methanol, aldehydes, and other organic impurities, which may potentially harm human health. While n-hexane is a low-toxicity solvent, its accumulation in the body may suppress the central nervous system, and long-term exposure can lead to polyneuropathy. Residues from the high-molecular-weight polyamide polymers and macroporous resins may cause allergic reactions, increase the burden on the kidneys, and cause kidney damage.

[0008] In summary, most publicly available methods for extracting dihydroquercetin from larch trees utilize high-concentration industrial ethanol. The organic solvents and materials used in the defatting and purification processes also have certain impacts on human health. Furthermore, there is no effective control over residual solvents and high-molecular-weight compounds remaining from the extraction process. Moreover, current methods of consuming dihydroquercetin also have drawbacks. Directly sublingually or orally administering high-purity dihydroquercetin compounds causes significant oral irritation, has an unpleasant taste, and is generally not widely accepted. Summary of the Invention

[0009] The purpose of this invention is to address the above-mentioned technical problems by providing a method for preparing a dihydroflavonoid polyphenol nutritional wine with a unique pine aroma, obtained by extracting dihydroflavonoid polyphenol nutrients during the pure grain liquor brewing process. This wine is safe for consumption and has a smooth taste. This invention achieves its purpose through the following technical solutions:

[0010] A method for preparing a pine-scented dihydroflavonoid polyphenol nutritional wine includes the following steps:

[0011] 1) Pine wood processing: Pine roots and / or pine branches are crushed, degreased, washed and dried to obtain pine wood chips for later use;

[0012] 2) Mixed ingredients: The brewing grains are mixed with pine sawdust to obtain mixed ingredients; wherein the pine sawdust accounts for 20% to 30% of the mass of the brewing grains;

[0013] 3) Soaking and steaming: Soak the mixed ingredients in water, drain them, add water again, and steam the mixture to obtain a moistened mixed brewing mixture;

[0014] 4) Enzyme inoculation: Spread the mixed brewing material to cool to 24-27℃, sprinkle in the yeast and combined enzymes, and ferment aerobically for 24-36 hours; the combined enzymes consist of cellulase, hemicellulase, β-glucosidase, xylanase and pectinase.

[0015] 5) Brewing and fermentation: After aerobic fermentation, the brewing materials undergo anaerobic fermentation for 30-40 days;

[0016] 6) Saturated steam fractionation: The fermented material (i.e., the wine lees) that has reached the end of fermentation is taken out and placed in a still, saturated steam is introduced to promote the transfer of extracts, and the liquor is fractionated, while the lees are retained except for the liquor; the lees are solid debris containing dihydroflavonoid polyphenol extracts.

[0017] 7) Extraction of dihydroflavonoid polyphenols:

[0018] S1 tank water separation: Add a 90℃~95℃ water bath to the lees after removing the alcohol, stir, filter at a temperature not lower than 80℃ to separate the lees from the water phase, collect the water phase and cool it to room temperature.

[0019] S2 acidification: Adjust the pH of the aqueous phase to 2.5–3.5;

[0020] S3 Low-temperature sedimentation: Place the aqueous phase at 2-5℃ and let it settle for 24 hours, then collect the precipitate;

[0021] S4 Rinsing and Acid Neutralization: Take ice water to rinse the precipitate several times until there is no turbidity, and stop rinsing when the pH value of the aqueous phase is 5-6.

[0022] S5 sedimentation and drying: Settle at 2-5℃ for 24 hours to separate the aqueous phase, collect the sediment and dry it to obtain an extract containing dihydroflavonoid polyphenols.

[0023] 8) Redissolution: Redissolve the extract obtained in step 7) into the fractionated liquor in step 6) to obtain the dihydroflavonoid polyphenol nutritional liquor and seal it in a ceramic jar in a cool place.

[0024] Further optimization involves using petroleum ether to degrease the pine wood in step 1); the length of the pine wood chips is 0.1–0.4 cm.

[0025] Furthermore, in step 2), the grains used for brewing are one or a mixture of several of sorghum, wheat, rice, and corn. Japonica sorghum grown at 46° North latitude is preferred.

[0026] Furthermore, in step 4), the quality of the yeast starter accounts for 6% to 12% of the quality of the grains used for brewing, and the enzyme activity is ≥10000u / g.

[0027] Furthermore, in step 4), the combined enzymes account for 2% to 4% of the total mass of the brewing grains; by mass, the combined enzymes comprise 40% to 50% cellulase, 30% to 40% hemicellulase, 10% to 12% β-glucosidase, 5% to 6% xylanase, and 5% to 6% pectinase; the cellulase has an enzyme activity ≥90,000 u / g, the hemicellulase has an enzyme activity ≥30,000 u / g, the β-glucosidase has an enzyme activity ≥100 u / g, the xylanase has an enzyme activity ≥50,000 u / g, and the pectinase has an enzyme activity ≥30,000 u / g.

[0028] Furthermore, in step 5), the temperature of the anaerobic fermentation of the brewing materials shall not exceed 25°C.

[0029] Furthermore, in step 6), the temperature of the saturated steam is not lower than 110°C.

[0030] Furthermore, in step 7), citric acid solution is used to adjust the pH value of the aqueous phase.

[0031] The advantages and beneficial effects of this invention are:

[0032] This invention uses larch wood chips as a raw material source for dihydroflavonoid polyphenols, which also serve as a breathable and porous auxiliary material in brewing. Based on the traditional solid-state fermentation method for brewing baijiu (Chinese white liquor), a combination enzyme, mainly cellulase, is added simultaneously with the yeast starter. During fermentation, the combination enzyme enzymatically hydrolyzes the cellulose in the pine wood chips, causing dihydroflavonoid polyphenols such as dihydroquercetin to gradually dissolve in the mash. The mash after removing the alcohol is separated and extracted to obtain an extract containing dihydroflavonoid polyphenols, which is then re-dissolved in the brewed baijiu.

[0033] This invention extracts dihydroflavonoid polyphenols, primarily dihydroquercetin, from pine wood during the brewing of baijiu (Chinese liquor). Compared to existing techniques that use high-concentration industrial ethanol to extract dihydroquercetin, this method leaves virtually no harmful solvent or substance residues. The extracted dihydroquercetin is highly safe for medicinal use, and the crude extract achieves a purity of over 90%. Furthermore, dihydroquercetin is generated simultaneously during the brewing process, eliminating the need for additional extraction equipment and solvents. Subsequent processing is also relatively simple, resulting in low investment, high efficiency, and strong practicality.

[0034] This invention simultaneously generates dihydroflavonoid polyphenols during the brewing process. The distilled lees also play a crucial role in adsorbing, filtering, and purifying the dihydroflavonoid polyphenol extract. Pine sawdust replaces rice husks, a brewing aid used for thousands of years, providing a breathable and porous material. The reconstituted liquor has a unique pine aroma. While drinking it, one can supplement with various dihydroflavonoid polyphenols, primarily dihydroquercetin, reducing the irritation and discomfort from direct oral intake, thus offering health benefits.

[0035] The combined enzymes in this invention not only degrade lignocellulose but also increase the utilization rate of brewing raw materials and improve the quality and taste of the wine. While the nutritional wine incorporates pine extracts during its preparation, resulting in a unique aroma due to its subtle pine fragrance, it often has an unpleasant, bitter taste that is not easily accepted. This combined enzyme, by adding a certain proportion of β-glucosidase, xylanase, and xylanase to the base of cellulase and hemicellulase, effectively regulates the taste of the nutritional wine, making it smooth on the palate, highly palatable, and with a slightly yellow and clear body.

[0036] The nutritional wine prepared by this invention was qualitatively analyzed by comparing it with standard references using a high-resolution liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument (MicrOTOF-QII). The analysis confirmed that it contains dihydroquercetin, hesperidin, pinocembrin, and other trace flavonoids, with dihydroquercetin accounting for more than 90%. Attached Figure Description

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0038] Figure 1 H+ m / z data for three dihydroflavonoid polyphenol compounds (source: ProteinScape™ database);

[0039] Figure 2 This is a high-resolution chromatogram and mass spectrum of the reference standard for quercetin (dihydroquercetin).

[0040] Figure 3 High-resolution chromatogram and mass spectrum of hesperidin reference standard;

[0041] Figure 4 High-resolution chromatogram and mass spectrum of pinocembrin reference standard;

[0042] Figure 5 Here is the high-resolution chromatogram and mass spectrum of the extract from Example 1;

[0043] Figure 6 Here is a high-resolution chromatogram / mass spectrum of the extract from Example 1 after further purification;

[0044] Figure 7 Here is the high-resolution chromatogram and mass spectrum of the nutritional wine from Example 1;

[0045] Figure 8 Here is the liquid chromatogram of the extract from Example 2;

[0046] Figure 9 The liquid chromatogram of the extracted recrystallized product in Example 2;

[0047] Figure 10 Here is the liquid chromatogram of the extract from Example 3;

[0048] Figure 11 Here is the liquid chromatogram of the extract from Example 4;

[0049] Figure 12 Here is the liquid chromatogram of the nutritional wine from Example 4;

[0050] Figure 13 This is a process flow diagram for Example 1. Detailed Implementation

[0051] The present invention will now be described in detail with reference to specific embodiments.

[0052] Example 1:

[0053] A method for preparing a pine-scented dihydroflavonoid polyphenol nutritional wine includes the following steps:

[0054] 1) Pine wood processing: Pine roots and / or pine branches are crushed, degreased, washed and dried to obtain pine wood chips for later use.

[0055] In this embodiment, the preferred pine sawdust consists of larch roots and branches from the Lesser Khingan Mountains region of Yichun. The collected tree roots are used as the main raw material for the extract; pruned branches can also be used. Roots are generally collected from about 50cm below ground level, while branches are collected with a diameter of 5cm or more. After being dried in a cool, shaded place, the thicker pine roots and branches (greater than 5cm) are split into smaller pieces, crushed into strips, and then transferred to a tumbler for debarking. While the tumbler is rolling, air is blown away to remove the bark and dust. Finally, a wood pulverizer is used to pulverize the wood into irregular sawdust approximately 0.25cm in length.

[0056] First, the pine sawdust is degreased by transferring it to a 304 stainless steel degreasing tank. Petroleum ether with a boiling range of 30-60℃ is injected and allowed to settle for approximately 30 minutes, with stirring during this period. The petroleum ether is then filtered out. The sawdust is spread evenly on a clean cement floor, and a gentle breeze of approximately 30℃ is circulated indoors to agitate and evaporate the petroleum ether. The filtered petroleum ether mixture is then recovered using a thin-film evaporator, and the concentrate is pine resin.

[0057] After degreasing, the sawdust is transferred to a washing tank, filled with purified water, and allowed to settle for 30-40 minutes with stirring. The water is then filtered out, and the sawdust is washed and placed on a tray in an infrared drying oven to dry to a moisture content of 10%-15% for later use.

[0058] 2) Mixing ingredients: The brewing grains and pine sawdust are mixed evenly to obtain the mixed ingredients; wherein the pine sawdust accounts for 20% to 30% of the mass of the brewing grains. In this embodiment, the brewing raw materials are selected from organically grown japonica sorghum varieties in Yichun area.

[0059] 3) Soaking and steaming: Soak the mixed ingredients in water, drain them, add water again, and then steam the mixture.

[0060] In this embodiment, the mixed ingredients are poured into a tank, filled with purified water, and soaked for 7-8 hours to achieve uniform water absorption and a moisture content of 35%-45%.

[0061] Drain the mixed ingredients and pour them into the still. Add an appropriate amount of purified water and steam for 20-40 minutes, then let it sit for 5-10 minutes, and finally steam for another 60 minutes.

[0062] 4) Enzyme Inoculation: In this embodiment, the re-steamed brewing material is removed from the still and spread on a clean cement floor. It is repeatedly stirred to quickly dissipate heat. After cooling to approximately 25°C, the yeast starter and combined enzymes are added, and the mixture is turned evenly. It is then transferred to the inoculation tank, covered with a breathable cotton cloth, and subjected to aerobic fermentation at approximately 25°C for 24–36 hours. The combined enzymes consist of cellulase, hemicellulase, β-glucosidase, xylanase, and pectinase. The yeast starter is sourced from the liquor powder of Jilin Changqinglong Group Co., Ltd., with an enzyme activity ≥10000 u / g. Specific ingredient components are shown in Table 1.

[0063] Table 1

[0064]

[0065] All enzymes used to degrade lignocellulose are food-grade.

[0066] The cellulase is a food-grade product from Shandong Longket Enzyme Preparation Co., Ltd., with an enzyme activity ≥90000u / g. It is used to enzymatically hydrolyze pine sawdust cellulose and brewing raw materials.

[0067] The hemicellulase is a food-grade product from Sichuan Huatang Jurui Biotechnology Co., Ltd., with an enzyme activity of ≥30000u / g. The hemicellulase accelerates the enzymatic hydrolysis of pine sawdust cellulose and also improves the fermentation rate of brewing.

[0068] The β-glucosidase is a food-grade product from Jiangsu Ruiyang Biotechnology Co., Ltd., with an enzyme activity ≥100u / g. The function of β-glucosidase is to assist cellulase in accelerating the hydrolysis of pine sawdust cellulose and reduce the inhibitory effect of the enzymatic hydrolysis process on cellulase.

[0069] Xylanase is a food-grade product from Sichuan Huatang Jurui Biotechnology Co., Ltd., with an enzyme activity ≥50000u / g. Xylanase assists cellulase in accelerating the hydrolysis of pine sawdust cellulose and promotes the dissolution of flavonoids in pine sawdust cellulose cells.

[0070] The pectinase is a food-grade product from Sichuan Huatang Jurui Biotechnology Co., Ltd., with an enzyme activity of ≥30000u / g. Pectinase assists cellulase in accelerating the hydrolysis of pine sawdust cellulose, destroying the cell wall structure of pine sawdust cellulose, promoting the dissolution of flavonoids in pine sawdust, and also increasing the clarity of brewed ethanol liquor.

[0071] 5) Brewing and fermentation: In this embodiment, the mixed material that has been cultured and fermented aerobically is cooled again to below 25°C, transferred to the fermentation pit, flattened and compacted, covered with a dense tarpaulin and sealed tightly. Anaerobic fermentation is carried out for 35 days. The fermentation time affects the production rate of ethanol liquor and also affects the enzymatic hydrolysis of pine sawdust and the extraction of flavonoid polyphenols.

[0072] 6) Saturated steam fractionation: After fermentation, the fermented material (also called the fermented liquor) is removed and placed in a still, through which saturated steam (approximately 110–120°C) is introduced to promote the transfer of extracts. The fractionated liquor with an ethanol content of 50–55% is collected by distillation. Distillation continues until the ethanol content of the distilled water is below 5% using an alcohol meter, at which point the steam supply is stopped. The distilled liquor has a faint pine aroma.

[0073] 7) Extraction of dihydroflavonoid polyphenols:

[0074] S1 Tank Water Separation: In this embodiment, the distilled lees are transferred to a 304 stainless steel tank, and an equal volume of hot purified water at 90℃~95℃ is added for mixing. The mixture is thoroughly stirred, and once the water temperature drops to approximately 80℃, a plate and frame filter is used to separate the lees from the aqueous phase. The aqueous phase is collected in a 304 stainless steel container and allowed to cool to room temperature. The lees also play a crucial role in adsorbing impurities during the aqueous phase separation process. These impurities include residues from the enzymatic hydrolysis of koji enzymes and cellulase, as well as other fusel alcohols generated during brewing.

[0075] S2 pH adjustment: The collected aqueous phase is adjusted to pH 2.5-3.5 using 1M food-grade citric acid solution.

[0076] S3 acidification and low-temperature sedimentation: After acidification, the aqueous phase is placed in an environment of 2-5℃ for sedimentation for 24 hours, and the precipitate is collected.

[0077] S4 Rinsing and Acid Neutralization: Take 10 times the volume of ice water at around 0°C, rinse the precipitate several times until there is no turbidity, and stop rinsing when the pH of the aqueous phase reaches 5-6.

[0078] S5 precipitation and acid neutralization: Allow to settle for 24 hours at 2-5℃. Separate the aqueous phase, collect the precipitate and place it in a desiccator. Dry with phosphorus pentoxide desiccant for 24 hours. The dried product is slightly yellow, yielding an extract containing dihydroflavonoid polyphenols.

[0079] 8) Redissolution: Redissolve the extract obtained in step 7) into the fractionated liquor in step 6) to obtain the dihydroflavonoid polyphenol nutritional liquor and seal it in a ceramic jar in a cool place.

[0080] In this embodiment, the extract from step 7) is taken and reconstituted at a concentration of 10 mg / ml of dihydroquercetin into a distilled liquor with an ethanol content of about 50% to prepare a dihydroflavonoid polyphenol nutritional liquor, which is then sealed in a ceramic jar in a cool place.

[0081] Alternatively, a low-ethanol-content (18%–22%) dihydroflavonoid polyphenol nutritional wine can be prepared. Take the extract from step 7), dissolve it in an appropriate amount of high-concentration white wine at a concentration of 5 mg / ml of dihydroquercetin, and then gradually expand the solution while warming until all the solution is dissolved. Seal the wine in a ceramic jar and store it in a cool place.

[0082] The following is the process for identifying the extract in step 7):

[0083] Purification and crystallization:

[0084] The extract was dissolved in 50%–55% distilled ethanol by heating it to a volume of 3, and then placed in a refrigerator at 2–5°C for 24 hours to crystallize. The crystals were filtered, collected, and dried in a desiccator with phosphorus pentoxide for 24 hours until the dried product was a slightly yellow, lumpy substance. The wet weight yield was approximately 3%.

[0085] Structural identification:

[0086] The structures of the extract and the repurified crystals were qualitatively analyzed using a Bruker high-resolution liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument, microTOF-QⅡ.

[0087] The following are the reference conditions for liquid chromatography-tandem mass spectrometry (LC-MS):

[0088] (1) Liquid chromatography-mass spectrometry conditions:

[0089] Column: C18, 100 mm × 2.1 mm; 1.7 μm

[0090] Mobile phase: 0.1% formic acid aqueous solution

[0091] Flow rate: 0.25 ml / min

[0092] Column temperature: 30℃

[0093] Injection volume: 2 μl

[0094] Preparation of reference solution:

[0095] Table 2

[0096]

[0097] Taxostatin (dihydroquercetin) reference solution: Weigh 10 mg of taxostatin reference standard into a 10 mL volumetric flask, add 0.1% formic acid-methanol to dissolve to the mark, and shake well to obtain the solution.

[0098] Hesperidin reference solution: Weigh 2.5 mg of hesperidin reference standard into a 10 ml volumetric flask, add 0.1% formic acid-methanol to dissolve to the mark, take 1 ml and put it into a 5 ml volumetric flask, add 0.1% formic acid-methanol to dissolve and dilute to the mark, and shake well to obtain the solution.

[0099] Pinus syringa reference solution: Weigh 2.5 mg of pinus syringa reference standard into a 10 ml volumetric flask, and dissolve to the mark with 0.1% formic acid-methanol. Take 1 ml of the solution and place it into a 5 ml volumetric flask, dissolve and dilute to the mark with 0.1% formic acid-methanol, and shake well.

[0100] Test solution: Weigh 10 mg of the extract and purified crystals into a 10 mL volumetric flask, add 0.1% formic acid-methanol to the mark, shake well and filter through a 0.45 μm microporous membrane before injection.

[0101] (2) Mass spectrometry conditions:

[0102] Scanning method: Positive and negative ion scanning

[0103] Scan start-end (mass-to-charge ratio): 50 m / z - 1200 m / z

[0104] Atomizer pressure: 0.6 Bar

[0105] Collision cell RF voltage: 100.0Vpp

[0106] Conclusion: The extract contains dihydroquercetin, hesperidin, pinocembrin, and other trace flavonoids, with dihydroquercetin being the main component. The high-resolution chromatogram and mass spectrum of the extract are shown below. Figure 5 The purified product contained over 97% dihydroquercetin, and its high-resolution chromatogram / mass spectrum is shown below. Figure 6 .

[0107] Referring to the data provided by the micrito-OTOF-QⅡ ProteinScape™ database, the m / z values ​​of the piperidine, hesperidin, and pinocembrin reference standards provided by Shanghai Yuanye Biotechnology Co., Ltd. were verified using the micrito-OTOF-QⅡ system. The results were consistent with the database data. The m / z verification table for the reference standards is shown in Table 3.

[0108] Table 3

[0109] Reference Standard Name Database m / z value Actual verification of m / z value Taxodia pine resin 305.0656 305.0657 hesperidin 289.0707 289.707 pinocin 257.0809 257.0826

[0110] Identification of the components of the nutritional wine in this embodiment:

[0111] The chromatographic and mass spectrometric data of the reference standards of piperidine, hesperidin, and pinocembrin from Shanghai Yuanye Biotechnology Co., Ltd. were verified and qualitatively identified using high-resolution liquid chromatography-mass spectrometry (HPLC-MS / MS) microTOF-QⅡ. The main components were dihydroquercetin, hesperidin, pinocembrin, and other trace flavonoids, with dihydroquercetin accounting for over 90%. The high-resolution chromatographic and mass spectra of the nutritional wine are shown below. Figure 7 .

[0112] Example 2

[0113] In this embodiment, the combined enzyme is adjusted to approximately 3.5% of the brewing raw material mass; other processes are the same as in Example 1. The ingredient composition is shown in Table 4.

[0114] Table 4

[0115]

[0116] The wet weight of the extract was approximately 760g. After drying in a desiccator with phosphorus pentoxide for 24 hours, the residue weighed approximately 630g. This residue was then ground in a mortar and pestle to form a slightly yellow collectible. The yield was approximately 3.2%.

[0117] Content detection by liquid chromatography

[0118] Liquid chromatography conditions:

[0119] Agilent 1260 High Performance Liquid Chromatography Diode Array Detector

[0120] Detection wavelength: 288nm

[0121] Chromatographic column: C18 column (250×4.0mm, 5μm)

[0122] Mobile phase: Methanol:Water: 40:60

[0123] Flow rate: 1.0 mL / min;

[0124] Injection volume: 10 μL;

[0125] Column temperature: 30℃;

[0126] The preparation of the reference solution and the test solution is the same as in Example 1.

[0127] Test results:

[0128] Liquid chromatography analysis showed that dihydroquercetin accounted for approximately 95% of the extract, while hesperidin and pinocembrin accounted for 2.1%–2.6%, respectively. (See details...) Figure 8 .

[0129] Recrystallization of the extract yielded over 97% dihydroquercetin. See details. Figure 9 .

[0130] Example 3

[0131] In this embodiment, the ratio of cellulase and hemicellulase in the ingredients was adjusted, and the mixed sawdust contained 20% pine branch sawdust. Other processes followed those in Example 1, and content detection followed those in Example 2. The ingredients are shown in Table 5.

[0132] Table 5

[0133]

[0134] Extract detection:

[0135] The mixture was obtained with a wet weight of approximately 700g. It was placed in a desiccator with phosphorus pentoxide as a desiccant and dried for 24 hours. The resulting product weighed 520g, with a yield of approximately 2.6%. The mixture was then ground into a slightly yellow powder using a mortar and pestle.

[0136] Liquid chromatography analysis showed that dihydroquercetin accounted for approximately 93% of the extract, while hesperidin and pinocembrin each accounted for approximately 2.7%–3.5%. (See details...) Figure 10 .

[0137] Example 4

[0138] This embodiment increases the amount of raw materials used; other processes follow the same procedure as in Example 1; and the extract analysis using liquid chromatography follows the same procedure as in Example 2. The ingredient composition is shown in Table 6.

[0139] Table 6

[0140]

[0141] Extract detection:

[0142] The mixture was obtained with a wet weight of approximately 2300g. It was placed in a desiccator with phosphorus pentoxide desiccant and dried for 24 hours. The weight was approximately 1900g, with a yield of about 2.2%. The mixture was then ground into a slightly yellow powder using a mortar and pestle.

[0143] Liquid chromatography analysis showed that dihydroquercetin accounted for approximately 93% of the extract, while hesperidin and pinocembrin each accounted for approximately 3.1%–3.6%. (See details...) Figure 11 .

[0144] The dried dihydroflavonoid polyphenols extracted in Example 4 were reconstituted with dihydroquercetin at a concentration of 10 mg / ml into a white wine containing approximately 50% ethanol that had been distilled, thus preparing a dihydroflavonoid polyphenol nutritional wine. The wine was then sealed in a ceramic jar in a cool, dark place.

[0145] Referring to Example 2, the content was determined by liquid chromatography. In the dihydroflavonoid polyphenol nutritional wine, dihydroquercetin accounted for 93.71%, hesperidin for 3.64%, and pinocembrin and other flavonoids for more than 2%. See details. Figure 12 .

[0146] In this embodiment of the invention, a bioavailability modeling comparison between dihydroflavonoid polyphenol nutritional wine and oral (sublingual) dihydroquercetin showed that the dihydroflavonoid polyphenol nutritional wine contains 5 mg / ml of dihydroquercetin (low-alcohol liquor), and 50 mg / 10 ml / time / day is approximately equivalent to 500 mg / time / day of sublingual dihydroquercetin, thus improving the bioavailability of dihydroquercetin.

[0147] Comparative experiment:

[0148] The study investigated the effect of the lignocellulose-degrading combination enzyme in the formulation on the extraction yield of dihydroflavonoid polyphenols. Referring to Example 2, the components were added at a ratio of 1 / 10, creating Example 2, which is a scaled-down version of Example 2. The experimental results were consistent with those of Example 2. A comparative example was set up based on Example 2. Fermentation was carried out in a 50L wooden fermentation tank, and saturated steam fractionation was performed using a 50L small high-pressure still.

[0149] Comparative Examples 1 to 6 are shown in Table 7. Other operations are the same as in Example 2.

[0150] Table 7

[0151]

[0152] The experimental results of Comparative Examples 1–6 are shown in Table 8.

[0153] Table 8

[0154]

[0155] As shown in Table 10, compared with Example 2, Comparative Example 1 did not add the combined enzyme, resulting in a significant decrease in extract yield and a strong pine flavor in the wine, making it less palatable. Comparative Example 2 significantly increased the content of cellulase and hemicellulase, but the total yield was not significantly higher than that of Example 2. Comparative Example 3 significantly decreased the content of cellulase and hemicellulase, resulting in a significant decrease in total yield compared to Example 2, but still higher than that of Comparative Example 1. In Comparative Example 4, the content of cellulase and hemicellulase remained unchanged compared to Example 2, but β-glucosidase was increased, resulting in no significant changes in yield and taste compared to Example 2. In Comparative Example 5, β-glucosidase was decreased, resulting in a decrease in total yield and a decline in taste and aroma compared to Example 2. In Comparative Example 6, β-glucosidase, xylanase, and pectinase were decreased, resulting in a significant decrease in yield and a decline in both taste and aroma compared to Example 2.

[0156] Comparative Examples 7-9 were prepared using the same feeding method as in Reference Example 2, with some changes to the process conditions (see Table 9). The experimental equipment used in Comparative Examples 1-6 was employed. Other operations were the same as in Example 1.

[0157] Table 9

[0158]

[0159] The experimental results of comparative examples 7–9 are shown in Table 10.

[0160] Table 10

[0161] Comparative Example Dihydroflavonoid polyphenol mixture Remark Example 1 3% wet weight before purification The baijiu has a smooth taste with a hint of pine. Comparative Example 7 The wet weight before purification is less than 1.5%. The taste of the baijiu was not significantly different from that in Example 1. Comparative Example 8 The wet weight before purification was 3.0–3.1%. The baijiu has a sour taste, and the yield is slightly improved. Comparative Example 9 The wet weight before purification is less than 1.5%. The taste of the baijiu was not significantly different from that in Example 1.

[0162] Comparative Examples 7 and 8, compared to Example 1, reduced and increased the anaerobic fermentation time, respectively. Reducing the fermentation time significantly decreased the wet weight of the extracted mixture, with little change in taste; while increasing the anaerobic fermentation time did not significantly improve the yield, resulted in a stronger sour taste, and a severe decline in taste. In Comparative Example 9, no saturated steam was introduced; atmospheric pressure boiling water steam fractionation had no significant impact on taste, but the wet weight of the mixture decreased significantly, and the extract was noticeably reduced.

[0163] Through the above comparative experiments, it can be confirmed that Examples 1 and 2 are the preferred experimental schemes of the present invention.

[0164] Finally, it should be noted that the above is only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the technology of the present invention.

Claims

1. A method for preparing a pine-scented dihydroflavonoid polyphenol nutritional wine, characterized in that: Includes the following steps: 1) Pine wood processing: Pine roots and / or pine branches are crushed, degreased, washed and dried to obtain pine wood chips for later use; 2) Mixed Ingredients: The brewing grains are mixed with pine sawdust to obtain the mixed ingredients; wherein the pine sawdust accounts for 20% to 30% of the mass of the brewing grains; 3) Soaking and steaming: Soak the mixed ingredients in water, drain them, add water again, and steam the mixture to obtain a moistened mixed brewing mixture; 4) Enzyme inoculation: Spread the mixed brewing material to cool to 24~27℃, sprinkle in the yeast and combined enzymes, and ferment aerobically for 24~36 hours; the combined enzymes consist of cellulase, hemicellulase, β-glucosidase, xylanase and pectinase; 5) Brewing and fermentation: After aerobic fermentation, the raw materials undergo anaerobic fermentation for 30-40 days; 6) Saturated steam fractionation: Take out the fermented material that has reached the end of fermentation and put it into a still, pass saturated steam through it, and fractionate the liquor, keeping the lees after removing the liquor; 7) Extraction of dihydroflavonoid polyphenols: S1 tank water separation: Add a 90℃~95℃ water bath to the lees after removing the alcohol, stir, filter at a temperature not lower than 80℃ to separate the lees from the water phase, collect the water phase and cool it to room temperature. S2 pH Adjustment: Adjust the pH of the aqueous phase to 2.5–3.5; S3 Low-temperature sedimentation: Place the aqueous phase at 2-5℃ and let it settle for 24 hours, then collect the precipitate; S4 Rinsing and acid neutralization: Take ice water to rinse the precipitate several times until there is no turbidity, and stop rinsing when the pH value of the aqueous phase is 5~6. S5 Sedimentation and drying: Settle at 2-5℃ for 24 hours to separate the aqueous phase, collect the sediment and dry it to obtain an extract containing dihydroflavonoid polyphenols. 8) Redissolution: Redissolve the extract obtained in step 7) into the fractionated liquor in step 6) to obtain the dihydroflavonoid polyphenol nutritional liquor and seal it in a ceramic jar in a cool place.

2. The method for preparing the pine wood-scented dihydroflavonoid polyphenol nutritional wine according to claim 1, characterized in that: In step 1), petroleum ether is used to degrease the pine wood; the length of the pine wood chips is 0.1~0.4cm.

3. The method for preparing the pine-scented dihydroflavonoid polyphenol nutritional wine according to claim 1, characterized in that: In step 2), the grains used for brewing are one or a mixture of sorghum, wheat, rice, and corn.

4. The method for preparing the pine-scented dihydroflavonoid polyphenol nutritional wine according to claim 1, characterized in that: In step 4), the quality of the yeast starter accounts for 6% to 12% of the quality of the grains used for brewing, and the enzyme activity is ≥10000u / g.

5. The method for preparing the pine-scented dihydroflavonoid polyphenol nutritional wine according to claim 1, characterized in that: In step 4), the combined enzymes account for 2% to 4% of the total mass of the brewing grains. By mass, the combined enzymes comprise 40% to 50% cellulase, 30% to 40% hemicellulase, 10% to 12% β-glucosidase, 5% to 6% xylanase, and 5% to 6% pectinase. The cellulase has an activity ≥90,000 u / g, the hemicellulase has an activity ≥30,000 u / g, the β-glucosidase has an activity ≥100 u / g, the xylanase has an activity ≥50,000 u / g, and the pectinase has an activity ≥30,000 u / g.

6. The method for preparing the pine-scented dihydroflavonoid polyphenol nutritional wine according to claim 1, characterized in that: In step 5), the temperature for anaerobic fermentation of the brewing materials should not exceed 25℃.

7. The method for preparing the pine wood-scented dihydroflavonoid polyphenol nutritional wine according to claim 1, characterized in that: In step 6), the temperature of the saturated steam shall not be lower than 110°C.

8. The method for preparing the pine-scented dihydroflavonoid polyphenol nutritional wine according to claim 1, characterized in that: In step 7), citric acid solution is used to adjust the pH of the aqueous phase.

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