Cereal beverage capable of dispelling effects of alcohol and protecting liver and preparation method of cereal beverage

Abstract

The invention belongs to the technical field of biology, and particularly relates to a cereal beverage capable of dispelling effects of alcohol and protecting liver and a preparation method thereof. Every 100 parts by weight of the cereal beverage comprises 5-25 parts by weight of freeze-dried powder of lalang grass rhizome-reed rhizome fermentation broth; 2-5 parts of a sweetening agent; 0.01 to 0.05 part of a stabilizing agent; 0.3 to 0.6 part of rice oil; 0.05 to 0.1 part of an emulsifier; the balance is rice milk; the lalang grass rhizome-reed rhizome fermentation liquor freeze-dried powder is prepared by mixing lalang grass rhizome leaching liquor and reed rhizome leaching liquor, inoculating lactobacillus plantarum WL05, fermenting, taking supernate and freeze-drying; the lactobacillus plantarum WL05 is preserved in the China General Microbiological Culture Collection Center on July 8, 2024, the preservation number is CGMCC No.31202, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Fermented freeze-dried powder obtained through fermentation of lactobacillus plantarum WL05, the green tea extract and the ampelopsis grossedentata extract are scientifically and reasonably matched, the functions of the fermented freeze-dried powder, the green tea extract and the ampelopsis grossedentata extract can be fully utilized, and compared with similar products, the ethanol degradation rate is high; and the preparation method is simple.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a hangover-relieving and liver-protecting cereal beverage and a preparation method thereof. Background Art

[0002] Alcohol is mainly metabolized in the liver because the liver has high levels of alcohol metabolizing enzymes. ADH and ALDH are two particularly important enzymes in the liver's ethanol metabolism process. About 90% of ethanol is oxidized into acetaldehyde by ADH, and acetaldehyde is then converted into acetic acid by ALDH and metabolized into carbon dioxide (CO2), fatty acids and water (H2O). Long-term excessive drinking can have harmful effects on the body, but drinking a large amount of alcohol at one time can also have toxic effects on many organs, among which the harm to the liver is the most significant, and it may also lead to the chronicity of alcoholic liver disease.

[0003] In recent years, with the improvement of people's material living standards, people have paid more and more attention to their own health. In order to effectively reduce the occurrence of alcohol-related diseases and alleviate the damage of alcohol to the liver, it is urgent to develop a product with medicinal and edible plants as raw materials. At present, although there are some alcohol sobering products on the market, such as alcohol sobering drugs, beverages and health products, most of the products have shortcomings in terms of the effect of solving the hangover problem, the safety of ingredients and side effects.

[0004] Therefore, it is necessary to develop a hangover and liver protection beverage to improve the hangover and liver protection effects. Summary of the invention

[0005] Based on the problems existing in the prior art, the present invention provides a hangover-relieving and liver-protecting cereal beverage and a preparation method thereof. The hangover-relieving and liver-protecting composition comprises three components: green tea extract, rattan tea extract, and lyophilized powder of Imperata rhizome-Phragmites plantarum WL05 fermentation broth. Through the synergistic effect of the three components, the rapid dispersion of alcohol can be promoted, the damage of alcohol to the liver can be reduced, and the effect of hangover-relieving and liver-protecting can be achieved.

[0006] The first aspect of the present invention is to provide a sobering and liver-protecting cereal drink, wherein every 100 parts by weight of the cereal drink comprises 5-25 parts of lyophilized powder of Imperata cylindrica-Phragmites australis fermentation liquid; 2-5 parts of sweetener; 0.01-0.05 parts of stabilizer; 0.3-0.6 parts of rice oil; 0.05-0.1 parts of emulsifier; and the balance is rice milk;

[0007] The cylindrical root-phragmites rhizome fermentation broth freeze-dried powder is prepared by mixing cylindrical root extract and phragmites rhizome extract, inoculating with Lactobacillus plantarum WL05, fermenting, and then freeze-drying the supernatant;

[0008] The plant lactobacillus WL05 was deposited in the China Center for Microbiological Culture Collection on July 8, 2024, with the deposit number: CGMCC No. 31202, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The freeze-dried powder obtained by co-fermentation of the Lactobacillus plantarum WL05 provided by the present invention and the Imperata cylindrica-Phragmites australis extract exhibits a strong ethanol degradation ability.

[0010] In some embodiments of the present invention, the cereal beverage also includes green tea extract and rattan tea extract; every 100 parts by weight of the cereal beverage includes 0.1-0.4 parts of green tea extract; 0.1-0.3 parts of rattan tea extract; 5-25 parts of freeze-dried powder of Imperata root-Phragmites australis fermentation broth; 2-5 parts of sweetener; 0.01-0.05 parts of stabilizer; 0.3-0.6 parts of rice oil; 0.05-0.1 parts of emulsifier; and the balance is rice milk.

[0011] Furthermore, the Lactobacillus plantarum WL05 was isolated from the feces of centenarians in a longevity village in Guangxi, and its nucleotide sequence is SEQ ID NO.1.

[0012] Further, the sweetener is selected from one or a combination of xylitol and erythritol;

[0013] In some preferred embodiments of the present invention, the sweetener is xylitol.

[0014] Furthermore, the stabilizer is selected from one or a combination of microcrystalline cellulose, carrageenan, and xanthan gum:

[0015] In some preferred embodiments of the present invention, the stabilizer is selected from microcrystalline cellulose and / or carrageenan;

[0016] Furthermore, the emulsifier is selected from one or a combination of monoglycerol fatty acid esters, diglycerol fatty acid esters, and polyglycerol fatty acid esters.

[0017] In some preferred embodiments of the present invention, the emulsifier is mono- and di-glycerol fatty acid esters;

[0018] In some preferred embodiments of the present invention, every 100 parts by weight of the cereal beverage includes the following amounts of each component: 0.2-0.4 parts of green tea extract; 0.1-0.3 parts of rattan tea extract; 5-25 parts of freeze-dried powder of Imperata rhizome - Phragmites australis plant fermentation liquid; 2-5 parts of xylitol, 0.01-0.03 parts of microcrystalline cellulose, 0.01-0.05 parts of carrageenan, 0.3-0.6 parts of rice oil, 0.05-0.1 parts of mono- and di-glycerol fatty acid esters, and the remainder is rice milk.

[0019] The second aspect of the present invention is to provide a method for preparing the alcohol-relieving and liver-protecting cereal beverage, comprising the following steps:

[0020] S1. Strain activation: Spread Lactobacillus plantarum WL05 on a plate culture medium for activation culture;

[0021] S2. Expanded culture: Pick the strain from the plate culture medium and place it in MRS liquid culture medium, and culture it at 30-37°C for 12-48h;

[0022] S3, preparation of rice milk: soak rice for 2-3h, grind with colloid mill, sieve and gelatinize, cool to below 50°C, add 0.1%-0.3% amylase, mix well, heat and enzymolysis, heat and kill enzyme, filter and set aside;

[0023] S4. Imperata root-Phragmites australis extract: crush Imperata root, add appropriate amount of water to extract, separate the solid and liquid, sterilize the liquid phase and set aside; crush Phragmites australis root, add appropriate amount of water to extract, separate the solid and liquid, sterilize the liquid phase and set aside; mix Imperata root extract and Phragmites australis extract according to proportion; inoculate Lactobacillus plantarum WL05 for fermentation.

[0024] S5. Preparation of lyophilized powder of Imperata rhizoma - Phragmites australis fermentation broth: taking the fermentation broth obtained in step S4, separating the solid from the liquid, pre-freezing the supernatant, and performing vacuum freeze drying on the pre-frozen sample to obtain lyophilized powder of Imperata rhizoma - Phragmites australis fermentation broth;

[0025] S6, preparation of cereal beverage: take the rice milk prepared in step S3, add green tea extract, rattan tea extract, and freeze-dried powder of Imperata rhizome-Phragmites australis plant fermentation liquid prepared in step S5, mix well, add stabilizer, sweetener, rice oil, emulsifier, homogenize, and sterilize to obtain the cereal beverage product.

[0026] Further, in step S3, the ratio of rice to water is 1:8-1:12; the gelatinization cooking time is not less than 40 minutes;

[0027] Furthermore, in step S3, the enzyme activity of the α-amylase used is not less than 3000 U / mL, and the dosage is 0.1% to 0.3% of the weight of the grain.

[0028] Further, in step S4, the ratio of Imperata cylindrica root extract to Phragmites australis root extract is 1:2-2:1;

[0029] Furthermore, in step S4, the fermentation time is 24-36 hours.

[0030] Furthermore, in step S4, the inoculation ratio of Lactobacillus plantarum WL05 is 3% to 9% of the mixed solution.

[0031] Furthermore, the viable count of Lactobacillus plantarum WL05 in step S4 is not less than 2×10 12 CFU / mL.

[0032] In some embodiments, in step S5, the pre-freezing step is specifically to take the supernatant and pre-freeze it at -20°C for about 12 hours, and then pre-freeze it at -80°C for about 4 hours.

[0033] In some embodiments, step S3, the selected and cleaned rice is mixed according to a rice-water ratio of 1:8-12 (w / w), the rice is soaked for 2-3 hours, the rice is ground with a colloid mill, passed through a 200-mesh sieve, gelatinized and steamed for 40 minutes, cooled to 50° C., 0.1%-0.3% of amylase is added, stirred and mixed evenly, kept at 50° C. for enzymolysis for 30 minutes, heated to 95° C. for enzyme inactivation for 20 minutes, filtered to obtain rice slurry for later use;

[0034] In some embodiments, step S4, preparation and fermentation of Imperata root-Phragmites rhizome extract: Take Imperata root and crush it with a grinder, weigh 100g, add 400g of deionized water, ultrasonically extract at 85℃ for 30min, filter twice through an 80-mesh sieve, sterilize in a water bath at 95℃ for 5min, and obtain sterile Imperata root slurry, which is cooled for standby use. Take Phragmites rhizome and crush it with a grinder, weigh 100g, add 400g of deionized water, ultrasonically extract at 85℃ for 30min, filter twice through an 80-mesh sieve, sterilize in a water bath at 95℃ for 5min, and obtain sterile Phragmites rhizome slurry, which is cooled for standby use. Mix Imperata root extract and Phragmites rhizome extract in a ratio of 1:2 for inoculation and culture; inoculate Lactobacillus plantarum WL05 in the Imperata root-Phragmites rhizome extract culture medium at a ratio of 3% to 9%, and ferment at 37℃ for 24-36h. The fermentation liquid obtained in the step was centrifuged at 3000 rpm and 4° C. for 15 min, the supernatant was pre-frozen in a -80° C. refrigerator overnight, and the pre-frozen sample was vacuum freeze-dried to obtain lyophilized powder of Imperata cylindrica-Phragmites australis fermentation liquid.

[0035] In some embodiments, in step S5, high pressure homogenization is performed for 5-10 minutes.

[0036] Beneficial effect: Compared with the prior art, the advantages of the present invention are at least that: it provides a hangover-relieving and liver-protecting cereal beverage, based on the high acid production capacity and vitality of Lactobacillus plantarum WL05, strong acid and bile salt resistance, and strong ethanol degradation ability, and further improves the ethanol degradation ability after culturing with Imperata rhizome-Phragmites australis matrix. The fermented freeze-dried powder obtained by fermentation of Lactobacillus plantarum WL05, as well as green tea extract and rattan tea extract are scientifically and reasonably compatible, which can fully utilize their functions and has a stronger ethanol degradation rate compared with similar products. The preparation method of the hangover-relieving and liver-protecting cereal beverage of the present invention is simple.

[0037] Biomaterial Deposit

[0038] Lactobacillus plantarum WL05 was deposited in the China Center for Microbiological Culture Collection on July 8, 2024, with the deposit number: CGMCC No.31202, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the electrophoresis diagram of Lactobacillus plantarum WL05 of the present invention (spotting order: WL01, WL04, WL05, WL06, WL07, WL08, WL09, WL010, Marker). DETAILED DESCRIPTION

[0040] The technical scheme of the present invention is described clearly and completely below in conjunction with the accompanying drawings and implementation cases. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and do not limit the claims of the present invention in any way. The experimental methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0041] Imperata root (also known as Imperata grass, Imperata grass, Imperata grass); purchased from: Ali Health Pharmacy;

[0042] Reed root (also known as: reed root, reed root, reed head, reed root); purchased from: Ali Health Pharmacy.

[0043] Related culture media and reagent formulas

[0044] MRS solid medium: peptone 10.0 g, sodium acetate trihydrate 3.0 g, beef extract 8.0 g, ammonium citrate trihydrate 2.0 g, yeast extract 4.0 g, magnesium sulfate heptahydrate 0.2 g, glucose 10.0 g, manganese sulfate monohydrate 0.04 g, Tween-80 1.0 mL, potassium hydrogen phosphate 2.0 g, agar 15 g, distilled water 1000 mL, adjust the pH to 6.4.

[0045] MRS liquid medium: peptone 10.0g, sodium acetate trihydrate 3.0g, beef extract 8.0g, ammonium citrate trihydrate 2.0g, yeast extract 4.0g, magnesium sulfate heptahydrate 0.2g, glucose 10.0g, manganese sulfate monohydrate 0.04g, Tween-80 1.0mL, potassium hydrogen phosphate 2.0g, distilled water 1000mL. Adjust pH to 6.4, sterilize at 121℃ for 15min, and cool for use.

[0046] MRS fermentation medium: peptone 10.0g, sodium acetate trihydrate 3.0g, beef extract 8.0g, ammonium citrate trihydrate 2.0g, yeast extract 4.0g, magnesium sulfate heptahydrate 0.2g, glucose 10.0g, manganese sulfate monohydrate 0.04g, Tween-80 1.0mL, potassium hydrogen phosphate 2.0g, 10% ethanol, distilled water 1000mL. Adjust pH to 6.4, sterilize at 121℃ for 15min, and cool for use.

[0047] Imperata rhizome-Phragmites australis extract matrix culture medium: Imperata rhizome raw material, at a material-water ratio of 1:4, ultrasonically extract at 85°C for 30 min, filter through 80-mesh sieve twice, sterilize at 95°C water bath for 5 min, and cool for use; Imperata rhizome raw material, at a material-water ratio of 1:4, ultrasonically extract at 85°C for 30 min, filter through 80-mesh sieve twice, sterilize at 95°C water bath for 5 min, and cool for use; Imperata rhizome extract and Phragmites australis extract are mixed at a ratio of 1:2 for inoculation and culture.

[0048] Skim milk matrix culture medium: 120 g skim milk powder, 1000 mL distilled water.

[0049] Method for determining ethanol tolerance: In the blank group, 1 mL of seed solution was inoculated into 50 mL of MRS liquid medium, cultured at 37°C for 12-24 hours, and the absorbance value Ac of the bacterial solution at 600 nm was determined. The experimental group was operated in the same way as the blank group, but the seed solution was inoculated into 50 mL of MRS fermentation medium, and the absorbance value was recorded as Ai. By comparing the absorbance values, the ethanol tolerance of the strain was determined. Calculation formula: Ethanol tolerance (%) = (Ai / Ac) × 100%.

[0050] Example 1 Isolation and Identification of Lactobacillus plantarum WL05

[0051] 1. Isolation of lactic acid bacteria

[0052] The lactic acid bacteria were isolated from the feces of the longevity elderly in the longevity village in Guangxi. The collected samples were diluted in gradients and inoculated into MRS solid medium. They were cultured at 37°C for 48 hours. Typical colonies on the plate were picked and streaked to obtain pure colonies. The pure colonies on the plate were picked into MRS liquid medium. After anaerobic culture at 37°C for 24 hours, the strain concentration reached 2.1-2.6×10 12 CFU / mL, refrigerate at 4℃ for later use or freeze-dried for storage.

[0053] 2. Identification of lactic acid bacteria

[0054] 16s rDNA sequence determination for strain identification: The prepared lactic acid bacteria genomic DNA was used as a template for PCR amplification, and PCR amplification was performed using a 50μL reaction system. Template DNA 2 uL, 1.5μL of 10mmol upstream and downstream primers, 25μL of 2×Taq enzyme, supplemented to 50μL with ddH2O. PCR cycle parameters are: 94℃ pre-denaturation for 5min; 94℃ denaturation for 1min, 64℃ annealing for 45s, 72℃ extension for 1min, 35 cycles; 72℃ extension for 8min. 5μL of PCR product was aspirated and mixed with 1mL Loading buffer, and electrophoresed with 1.0% agarose gel at a voltage of 80V. After the electrophoresis, it was stained with ethidium bromide EB for 20-30min and photographed. After electrophoresis detection of the amplified product, it was sent to Shanghai Shenggong Bioengineering Technology Service Co., Ltd. for sequencing. The sequence obtained by sequencing was compared with the sequence in the GenBank database, and the results showed that the homology between strain WL05 and Lactobacillus plantarum was 99.9%. The results showed that the strain WL05 of the present invention is Lactobacillus plantarum (see SEQ ID NO.1 for the sequence listing).

[0055] 3. Determination of acid production and ethanol tolerance of Lactobacillus plantarum WL05

[0056] Acid production capacity of lactic acid bacteria: Take the strains after refrigeration in Example 1 (live bacteria concentration 2.1-2.6×10 12 CFU / mL), inoculated into MRS liquid medium at a ratio of 3% (v / v), cultured at 37°C for 24h, and the pH of the fermentation broth was measured with a pH meter. Each strain was measured in parallel 3 times, and the results were averaged. The results showed that the pH value of the fermentation broth of Lactobacillus plantarum WL05 after 24h of fermentation in MRS matrix was 3.59, indicating that the strain had good acid production ability and good growth condition.

[0057] After the plant lactobacillus WL05 was fermented in MRS medium at 37°C for 24 hours, the pH value of the fermentation liquid was 3.59, and the number of viable bacteria was greater than 2.1×10 12 CFU / mL, it can be seen that Lactobacillus plantarum WL05 has higher acid production ability and vitality.

[0058] The strains after refrigeration in Example 1 were tested for their ethanol resistance: the results showed that the ethanol resistance of Lactobacillus plantarum WL05 was 80.5%, which showed a high ethanol tolerance.

[0059] 4. Determination of ethanol degradation rate

[0060] (1) Preparation of ethanol standard curve: add 0.1 mL of ethanol standard solution with volume concentration of 0.1%, 0.2%, 0.4%, 0.6%, 0.8% and 1.0% (v / v) to 10 mL culture tubes respectively, add 1 mL of potassium dichromate-sulfuric acid solution to each test tube, and shake well; place the test tube in a boiling water bath for 10 min, and measure the absorbance of potassium dichromate-sulfuric acid solution at a wavelength of 610 nm; prepare the ethanol standard curve with ethanol volume concentration (v / v) as the horizontal axis and absorbance as the vertical axis.

[0061] Among them, ethanol standard solution: take an appropriate amount of anhydrous ethanol, add it into sterile water to prepare ethanol standard solutions of different concentrations (0.1%, 0.2%, 0.4%, 0.6%, 0.8% and 1.0% (v / v)); potassium dichromate-sulfuric acid solution: take 1g of potassium dichromate and dissolve it in 25mL of water, and slowly add 4mL of concentrated sulfuric acid while stirring.

[0062] (2) Determination of ethanol degradation ability: Lactobacillus plantarum activated twice was inoculated into MRS liquid enrichment medium for enrichment for 5-10 hours, then diluted and spread on solid screening medium. A single colony was picked from the screening medium and inoculated into MRS liquid medium for 18 hours, and the OD was measured. The bacterial solution was adjusted to 10 7 CFU / mL. Inoculate at 5% inoculation volume into MRS ethanol culture medium of different concentrations (5%, 10%, 15%), and use MRS ethanol culture medium without bacterial solution as control, and culture for 24h.

[0063] Centrifuge the 24h fermentation culture (10000rpm, 10min), take 0.1ml of the supernatant, add 1mL of potassium dichromate-sulfuric acid solution, and shake well; place the test tube in a boiling water bath for 10min and measure the absorbance of the potassium dichromate-sulfuric acid solution at a wavelength of 610nm.

[0064] The ethanol residual amount is calculated using the ethanol standard curve, and the quantitative size of the strain's alcohol sobering ability can be obtained by comparison.

[0065] Lactobacillus plantarum WL05 was fermented in MRS medium containing 10% ethanol concentration at 37°C for 19-24h, and the ethanol degradation rate was measured to be 42.5%. Subsequently, the fermentation liquid was inoculated into skim milk matrix medium and Imperata root-Phragmites australis matrix medium at a 6% inoculum amount, respectively. After fermentation in skim milk matrix medium at 37°C for 24h, the ethanol degradation rate was measured to be 35.8%, and after fermentation in Imperata root-Phragmites australis matrix medium at 37°C for 24h, the ethanol degradation rate was measured to be 63.9%.

[0066] Lactobacillus plantarum WL05 was fermented at 37°C for 19-24h in MRS medium containing 15% ethanol concentration, and the ethanol degradation rate was measured to be 40.2%. Subsequently, the fermentation liquid was inoculated into skim milk matrix medium and Imperata root-Phragmites australis matrix medium according to 6% inoculation amount, and the ethanol degradation rate was measured to be 32.5% after fermentation at 37°C in skim milk matrix medium for 24h, and the ethanol degradation rate was measured to be 60.4% after fermentation at 37°C in Imperata root-Phragmites australis matrix medium for 24h.

[0067] 5. Alcohol Dehydrogenase Activity Assay

[0068] Preparation of fermentation broth

[0069] (1) Preparation of bacterial solution: Inoculate the bacterial strain into MRS liquid culture medium and culture at 37°C for 12-24 h.

[0070] (2) Fermentation to produce ethanol dehydrogenase: Add the test bacterial solution to 10 mL of MRS medium, skimmed milk medium or Imperata cylindrica-Phragmites australis medium at a volume ratio of 6%. After culturing in a constant temperature incubator at 37°C for 24-60 hours, centrifuge at 4000 rpm for 10 minutes and collect the supernatant fermentation solution.

[0071] Determination of alcohol dehydrogenase in fermentation broth: Referring to Chai Zheng, the modified Waller-Hoch method was used to detect ADH activity. 1.5 mL of pH 8.8 sodium pyrophosphate solution was added to a stoppered test tube, followed by 1.0 mL of 0.027 mol / L oxidized coenzyme I (NAD + ), 0.5mL 11% ethanol solution and 0.1mL sample solution. Mix well and place in a 37℃ water bath for 5min. After the insulation, add 0.1mL ADH (0.25U / mL) and replace the 11.5% ethanol solution with 0.5mL distilled water for blank control zeroing. Then shake the test tube solution, set the 0μg / mL experimental group as the negative control, shake the solution and then measure the absorbance value A340 at a wavelength of 340nm. The change in absorbance here can represent the enzymatic reaction rate of ADH. Record once every 30s and continuously record for 5min, and calculate the average absorbance value per minute. The specific calculation is shown in formulas (1) and (2):

[0072]

[0073] Where: V is the total reaction volume; M is the sample amount; ΔA340 / min is the average absorbance per minute.

[0074] Activation rate / % = (U1-U0) / U0×100% (2)

[0075] Where: U1 and U0 are the enzyme activities of the experimental group and the control group respectively. The results were measured in parallel for 3 times.

[0076] The alcohol dehydrogenase activity of Lactobacillus plantarum WL05 was detected to be over 15U / mL in the fermentation broth of MRS medium, over 10U / mL in the fermentation broth of skimmed milk matrix medium, and over 63U / mL in the fermentation broth of Imperata root-Phragmites australis matrix medium.

[0077] In MRS medium containing 10% ethanol, after fermentation at 37℃ for 19-24 hours, the activation rate of alcohol dehydrogenase in the fermentation broth of Lactobacillus plantarum WL05 was 43.53%. Subsequently, the fermentation broth was inoculated into skim milk matrix medium and Imperata cylindrica-Phragmites australis matrix medium, respectively. After fermentation at 37℃ for 24 hours in skim milk matrix medium, the activation rate of alcohol dehydrogenase in the fermentation broth was 32.26%; while after fermentation at 37℃ for 24 hours in Imperata cylindrica-Phragmites australis matrix medium, the activation rate of alcohol dehydrogenase in the fermentation broth was 66.5%.

[0078] In MRS medium containing 15% ethanol, Lactobacillus plantarum WL05 was fermented at 37°C for 19-24 hours, and the activation rate of alcohol dehydrogenase in the fermentation broth was 41.2%. Subsequently, the fermentation broth was inoculated into skim milk matrix medium and Imperata cylindrica-Phragmites australis matrix medium, respectively. After fermentation at 37°C for 24 hours in skim milk matrix medium, the activation rate of alcohol dehydrogenase was 30.6%, while after fermentation at 37°C for 24 hours in Imperata cylindrica-Phragmites australis matrix medium, the activation rate of alcohol dehydrogenase in the fermentation broth was 62.6%.

[0079] 6. Determination of acid and bile resistance of Lactobacillus plantarum WL05

[0080] The Lactobacillus plantarum WL05 was spread on a plate culture medium for activation culture, and then the strain was picked from the plate culture medium and placed in MRS liquid culture medium, and cultured at 37°C for 12-24h;

[0081] The Lactobacillus plantarum strain WL05 culture was centrifuged at 10000×g for 5 min to collect the cells, and washed twice with PBS, resuspended with physiological saline, and 1 mL of the bacterial suspension was inoculated into 9 mL of simulated gastric fluid at pH 3.0, shaken evenly, and cultured in a 37°C incubator. Samples were taken at 0 h and 3 h to plate, and the number of viable cells was determined, and the survival rate was calculated;

[0082] The culture of Lactobacillus plantarum strain WL05 was centrifuged at 10000×g for 5 min to collect the cells, washed twice with PBS, resuspended with physiological saline, and 1 mL of the bacterial suspension was inoculated into MRS liquid culture medium containing 0%, 0.1%, 0.3% and 0.5% (w / v) bile salts, and cultured at 37°C for 3 h. Samples were taken and plated at 0 h and 3 h to determine the number of viable cells and calculate the survival rate.

[0083] The results showed that the survival rate of Lactobacillus plantarum WL05 in artificial gastric juice with a pH of 3.0 was 69.2%, and the survival rates after culturing for 3 hours in MRS medium with bile salt concentrations of 0.10%, 0.30%, and 0.50% were 74.5%, 43.7%, and 15.5%, respectively.

[0084] 7. Determination of Antioxidant Capacity of Lactobacillus plantarum WL05

[0085] (1) DPPH clearance determination

[0086] The Lactobacillus plantarum WL05 was spread on a plate culture medium for activation culture, and then the strain was picked from the plate culture medium and placed in MRS liquid culture medium, and cultured at 37° C. for 12-24 hours; the Lactobacillus plantarum strain WL05 culture was centrifuged at 10000×g for 5 minutes, and the supernatant was taken as a sample.

[0087] Accurately measure 0.5 mL of sample solution and add it to a stoppered test tube. Then add 0.2 mL of DPPH solution (0.2 mmol / LDPPH dissolved in 95% ethanol solution). Mix well and react at 40°C in the dark for 20 minutes. Measure the absorbance at a wavelength of 517 nm and record it as A. i Blank group: Accurately measure 0.5 ml of sample solution and add it to a stoppered test tube. Replace the DPPH solution with 0.2 ml of 95% ethanol solution. After mixing, measure the absorbance at a wavelength of 517 nm and record it as A. j ; Control group: Add 2.0mL DPPH solution and 2mL 95% ethanol solution to a stoppered test tube, and measure the absorbance at a wavelength of 517nm, which is recorded as A c .

[0088] The total reaction volume of the above experimental groups was 4 mL, which was supplemented with 95% ethanol solution. The DPPH free radical scavenging rate was calculated according to the following formula: Scavenging rate (%): = [1-(A i -A j )] / A c *100

[0089] The results showed that the DPPH scavenging rate of Lactobacillus plantarum WL05 was 78.5%.

[0090] (2) Determination of reducing ability

[0091] The Lactobacillus plantarum WL05 was spread on a plate culture medium for activation culture, and then the strain was picked from the plate culture medium and placed in MRS liquid culture medium, and cultured at 37° C. for 12-24 hours; the Lactobacillus plantarum strain WL05 culture was centrifuged at 10000×g for 5 minutes, and the supernatant was taken as a sample.

[0092] Take 1.0mL of sample in a centrifuge tube, add 2.5mL of 0.2mol / L phosphate buffer solution (pH6.6) and 1% potassium ferricyanide solution, mix thoroughly, keep warm in a water bath at 50℃ for 20min, quickly cool to room temperature, add 2.5mL of 10% trichloroacetic acid solution, centrifuge at 3500r / min for 10min, accurately pipette 2.5ml of supernatant into a stoppered test tube, add 2.5mL of distilled water and 0.5mL of 0.1% ferric chloride solution in sequence, mix thoroughly, and measure the absorbance of the solution at a wavelength of 200nm. VC is used as the control sample.

[0093] The results showed that the reducing capacity of Lactobacillus plantarum WL05 reached 0.85, compared with the reducing capacity of VC (1.20), indicating that Lactobacillus plantarum WL05 has high antioxidant activity.

[0094] 8. Determination of antibacterial ability of Lactobacillus plantarum WL05

[0095] Lactobacillus plantarum WL05 was inoculated into the culture medium and cultured at 37°C for 24 hours. The antibacterial ability of lactic acid bacteria against Escherichia coli, Staphylococcus aureus, Salmonella, and Bacillus subtilis was determined by the Oxford cup method. The bacterial suspensions of Escherichia coli, Staphylococcus aureus, Salmonella, and Bacillus subtilis were spread into LB culture medium, 200 μL of lactic acid bacteria fermentation liquid was added to the Oxford cup, and cultured at a constant temperature for 48 hours, and the inhibition zone was observed.

[0096] The results showed that Lactobacillus plantarum WL05 had high antibacterial ability against Escherichia coli, Staphylococcus aureus, Salmonella and Bacillus subtilis, and the diameters of the inhibition zones were 22.50 mm, 18.35 mm, 17.22 mm and 16.50 mm, respectively.

[0097] Example 2 Preparation of a Cereal Beverage for Relieving Alcohol and Protecting Liver

[0098] (1) Preparation of cereal pulp

[0099] The selected and cleaned rice was mixed in a rice-water ratio of 1:12 (w / w), the rice was soaked for 2-3 hours, the rice was ground with a colloid mill, passed through a 200-mesh sieve, gelatinized and steamed for 40 minutes, cooled to 50°C, 0.3% amylase was added, stirred and mixed evenly, kept at 50°C for enzymolysis for 30 minutes, heated to 95°C for enzyme inactivation for 20 minutes, filtered and set aside;

[0100] (2) Imperata cylindrica-Phragmites australis extract

[0101] Take Imperata root, grind it with a grinder, weigh 100g, add 400g deionized water, ultrasonically extract at 85℃ for 30min, filter through 80-mesh sieve twice, sterilize in 95℃ water bath for 5min, and then obtain sterile Imperata root slurry, which is cooled for use. Take Phragmites australis, grind it with a grinder, weigh 100g, add 400g deionized water, ultrasonically extract at 85℃ for 30min, filter through 80-mesh sieve twice, sterilize in 95℃ water bath for 5min, and then obtain sterile Phragmites australis slurry, which is cooled for use. Mix Imperata root extract and Phragmites australis extract in a ratio of 1:2 for inoculation and culture;

[0102] (3) Fermented Imperata cylindrica-Phragmites australis extract

[0103] The Imperata rhizome-Phragmites australis extract medium was inoculated with Lactobacillus plantarum WL05 at a ratio of 3%, and fermented at 37°C for 24-36 hours.

[0104] (4) Preparation of freeze-dried powder of Imperata cylindrica-Phragmites australis fermentation broth

[0105] The fermentation liquid obtained in step (3) was centrifuged at 3000 rpm and 4°C for 15 min, the supernatant was placed in a -80°C refrigerator for pre-freezing overnight, and the pre-frozen sample was vacuum freeze-dried to obtain a lyophilized powder of Imperata cylindrica-Phragmites australis fermentation liquid;

[0106] (5) Cereal Beverage Preparation

[0107] The weight of each component of the present embodiment is calculated based on a total amount of 100g: the dosage of each component is as follows: add 100mg of rattan tea extract, add 5g of white imperata root-reed root plant fermentation liquid freeze-dried powder, add 100, 200, 300, and 400mg of green tea extract respectively, after the materials are mixed evenly, add 2g of xylitol, 0.01g of microcrystalline cellulose, 0.02g of carrageenan, 0.6g of rice oil, 0.1g of mono- and diglycerol fatty acid esters, and the remainder is rice milk, stir the mixture evenly, perform homogenization, homogenize for 5-10min, can, and sterilize to obtain a cereal beverage product.

[0108] Table 1

[0109]

[0110]

[0111] Example 3 Preparation of a Cereal Beverage for Relieving Alcohol and Protecting Liver

[0112] The preparation method of the cereal beverage in this embodiment is substantially the same as that in Embodiment 2. In the step (1) of preparing the cereal slurry, rice slurry is prepared according to a rice-water ratio of 1:8 (w / w), and 0.1% amylase is added;

[0113] In step (3) of preparing the cereal beverage, 9% Lactobacillus plantarum WL05 is inoculated into the Imperata cylindrica-Phragmites australis extract medium;

[0114] In step (5) of preparing the cereal beverage, the formulations of the components are slightly different;

[0115] The weight of each component of this embodiment is calculated based on a total amount of 100g: the dosage of each component is as follows: add 400mg green tea extract, add 5g of white imperata root-reed root plant fermentation liquid freeze-dried powder, add 100, 200, and 300 of rattan tea extract respectively, after the materials are mixed evenly, add 4g of xylitol, 0.02g of microcrystalline cellulose, 0.03g of carrageenan, 0.5g of rice oil, 0.08g of mono- and diglycerol fatty acid esters, and the remainder is rice milk, stir the mixture evenly, perform homogenization, homogenize for 5-10min, can, and sterilize to obtain a cereal beverage product.

[0116] Table 2

[0117] Rattan Tea Extract Ethanol degradation rate % 200mg 66.9％ 300mg 64.6％

[0118] Example 4 Preparation of a Cereal Beverage for Relieving Alcohol and Protecting Liver

[0119] The preparation method of the cereal beverage in this embodiment is substantially the same as that in Embodiment 2, except that in the step (1) of preparing the cereal slurry, rice slurry is prepared according to a rice-water ratio of 1:10 (w / w), and 0.2% amylase is added;

[0120] In step (3) of preparing the cereal beverage, 6% Lactobacillus plantarum WL05 is inoculated into the Imperata cylindrica-Phragmites australis extract medium;

[0121] In step (5) of preparing the cereal beverage, the formulations of the components are slightly different;

[0122] The weight of each component of the present embodiment is calculated based on a total amount of 100g: the dosage of each component is as follows: add 400mg green tea extract, add 200mg rattan tea extract, add 5, 15, and 25g of white imperata root-reed root plant fermentation liquid freeze-dried powder materials respectively, mix them evenly, add 5g of xylitol, 0.03g of microcrystalline cellulose, 0.05g of carrageenan, 0.3g of rice oil, 0.05g of mono- and diglycerol fatty acid esters, and the remainder is rice milk. Stir the mixture evenly, perform homogenization, homogenize for 5-10min, can, and sterilize to obtain a cereal beverage product.

[0123] Table 3

[0124]

[0125]

[0126] Example 5 Preparation of a Cereal Beverage for Relieving Alcohol and Protecting Liver

[0127] The method for preparing the cereal beverage in this embodiment is substantially the same as that in Embodiment 4, except that in step (5) the cereal beverage preparation step, the formula of each component is slightly different;

[0128] The weight of each component of the present embodiment is calculated based on a total amount of 100g: the dosage of each component is as follows: add 0 / 400mg green tea extract, add 0, 100, 200mg rattan tea extract, add 5, 15, 25g of white cypress root-reed root plant fermentation liquid freeze-dried powder respectively, mix the materials evenly, add 5g of xylitol, 0.03g of microcrystalline cellulose, 0.05g of carrageenan, 0.3g of rice oil, 0.05g of mono- and di-glycerol fatty acid esters, and the remainder is rice milk. Stir the mixture evenly, perform homogenization, homogenize for 5-10min, can, and sterilize to obtain a cereal beverage product.

[0129] Table 4

[0130]

[0131] From the data in Table 4, it can be seen that adding the lyophilized powder of Imperata cylindrica-Phragmites australis plant fermentation liquid to the beverage alone can achieve a higher ethanol degradation rate, and the ethanol degradation rate can be further improved after compounding with green tea extract and rattan tea extract.

[0132] Example 10 Comparative experiment of alcohol sobering and liver protecting cereal beverage and commercially available alcohol sobering products

[0133] Comparative experiment on direct degradation of ethanol

[0134] (1) Preparation of ethanol standard curve

[0135] To a 50mL volumetric flask, add 1mL of ethanol standard solution with volume concentration of 10%, 20%, 30%, 40%, 50%, and 60% (v / v), respectively, make up to volume, take 1mL of ethanol with different volume concentrations into test tubes, add 5mL of potassium dichromate and 2.5mL of concentrated sulfuric acid to each test tube, and shake well; measure the absorbance of potassium dichromate-sulfuric acid solution at a wavelength of 610nm; use the volume concentration of ethanol (v / v) as the horizontal coordinate and the absorbance value as the vertical coordinate to prepare an ethanol standard curve.

[0136] In order to evaluate the hangover-relieving ability of the prepared cereal beverage, an ethanol degradation experiment was carried out. 50° commercially available liquor (ethanol content of about 50%) was used as a substrate for adding reaction components of the hangover-relieving experiment. Commercially available hangover-relieving products (Kendixing, Sweishi Qingglycosides, Hovenia dulcis, artichoke plant beverage, Xanthoceras sorbifolia Leaf Vine Tea Turmeric Plant Beverage, Nanjing Tongrentang Green Gold Home Pueraria lobata Turmeric Hovenia dulcis beverage) were used as parallel controls. Only 50° liquor was added as a blank control. There were 3 parallels in each group, and the degradation rate was calculated based on the standard curve.

[0137] Table 5

[0138] product Ethanol degradation rate % Cereal beverage for hangover and liver protection 70.5％ Kendi wakes up 41.5％ Xanthoceras sorbifolia leaf vine tea turmeric plant drink 25.7％ Sweishi Qingglycosides Hovenia dulcis Artichoke Plant Drink 50.5％ Nanjing Tongrentang Green Gold Garden Pueraria Turmeric Hovenia Dulcis Drink 69.2％

[0139] It can be seen from the data in Table 5 that the hangover-relieving and liver-protecting cereal beverage prepared by the present invention has a higher ethanol degradation rate than similar products currently available on the market, and can achieve a better hangover-relieving and liver-protecting effect.

[0140] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A grain drink for sobering up and protecting liver, characterized in that: Every 100 parts by weight of the cereal beverage includes 5-25 parts of lyophilized powder of Imperata cylindrica-Phragmites australis fermented liquid; 2-5 parts of sweetener; 0.01-0.05 parts of stabilizer; 0.3-0.6 parts of rice oil; 0.05-0.1 parts of emulsifier; and the balance is rice milk; The cylindrical root-phragmites rhizome fermented liquid freeze-dried powder is prepared by mixing cylindrical root extract and phragmites rhizome extract, inoculating with Lactobacillus plantarum WL05, fermenting, and then freeze-drying the supernatant; The plant lactobacillus WL05 was deposited in the China Center for Microbiological Culture Collection on July 8, 2024, with the deposit number: CGMCC No.31202, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. The alcohol sobering and liver protecting cereal beverage according to claim 1, characterized in that: The cereal beverage also includes green tea extract and rattan tea extract; Every 100 parts by weight of the cereal beverage includes: 0.1-0.4 parts of green tea extract; 0.1-0.3 parts of rattan tea extract; 5-25 parts of lyophilized powder of Imperata root and Phragmites australis fermentation liquid; 2-5 parts of sweetener; 0.01-0.05 parts of stabilizer; 0.3-0.6 parts of rice oil; 0.05-0.1 parts of emulsifier; and the balance is rice milk.

3. The alcohol sobering and liver protecting cereal beverage according to claim 1, characterized in that: The Lactobacillus plantarum WL05 is isolated from the feces of centenarians in a longevity village in Guangxi, and its nucleotide sequence is SEQ ID NO.

1.

4. The alcohol sobering and liver protecting cereal beverage according to claim 1, characterized in that: The sweetener is selected from one or a combination of xylitol and erythritol; The stabilizer is selected from one or a combination of microcrystalline cellulose, carrageenan, and xanthan gum: The emulsifier is selected from one or a combination of monoglycerol fatty acid esters, diglycerol fatty acid esters and polyglycerol fatty acid esters.

5. The hangover-relieving and liver-protecting cereal beverage according to claim 4, characterized in that: Every 100 parts by weight of the cereal drink includes the following components: 0.2-0.4 parts of green tea extract; 0.1-0.3 parts of rattan tea extract; 5-25 parts of freeze-dried powder of Imperata cylindrica-Phragmites australis plant fermentation liquid; 2-5 parts of xylitol, 0.01-0.03 parts of microcrystalline cellulose, 0.01-0.05 parts of carrageenan, 0.3-0.6 parts of rice oil, 0.05-0.1 parts of mono- and di-glycerol fatty acid esters, and the remainder is rice milk.

6. The method for preparing the hangover-relief and liver-protecting cereal beverage according to any one of claims 1 to 5, characterized in that: The steps include: S1. Strain activation: Spread Lactobacillus plantarum WL05 on a plate culture medium for activation culture; S2. Expanded culture: Pick the strain from the plate culture medium and place it in MRS liquid culture medium, and culture it at 37°C for 12-48h; S3, preparation of rice milk: soak rice for 2-3h, grind with colloid mill, sieve and gelatinize, cool to below 50°C, add 0.1%-0.3% amylase, mix well, heat and enzymolysis, heat and kill enzyme, filter and set aside; S4. Imperata root-Phragmites australis extract: crush Imperata root, add appropriate amount of water to extract, separate the solid and liquid, sterilize the liquid phase and set aside; crush Phragmites australis root, add appropriate amount of water to extract, separate the solid and liquid, sterilize the liquid phase and set aside; mix Imperata root extract and Phragmites australis extract according to proportion; inoculate Lactobacillus plantarum WL05 for fermentation. S5. Preparation of lyophilized powder of Imperata rhizoma - Phragmites australis fermentation broth: taking the fermentation broth obtained in step S4, separating the solid from the liquid, pre-freezing the supernatant, and performing vacuum freeze drying on the pre-frozen sample to obtain lyophilized powder of Imperata rhizoma - Phragmites australis fermentation broth; S6, preparation of cereal beverage: take the rice milk prepared in step S3, add green tea extract, rattan tea extract, and freeze-dried powder of Imperata rhizome-Phragmites australis plant fermentation liquid prepared in step S5, mix well, add stabilizer, sweetener, rice oil, emulsifier, homogenize, and sterilize to obtain the cereal beverage product.

7. The method for preparing the hangover-relieving and liver-protecting cereal beverage according to claim 6, characterized in that: In step S3, the rice-water ratio is 1:8-1:12; the gelatinization cooking time is not less than 40 minutes; In step S3, the enzyme activity of the α-amylase used is not less than 3000 U / mL, and the dosage is 0.1% to 0.3% of the weight of the grain.

8. The method for preparing the hangover-relieving and liver-protecting cereal beverage according to claim 6, characterized in that: In step S4, the ratio of Imperata cylindrica root extract to Phragmites australis root extract is 1:2-2:1; In step S4, the fermentation time is 24-36 hours.

9. The method for preparing the hangover-relieving and liver-protecting cereal beverage according to claim 6, characterized in that: In step S4, the inoculation ratio of Lactobacillus plantarum WL05 is 3% to 9% of the mixed solution.

10. The method for preparing the hangover-relieving and liver-protecting cereal beverage according to claim 6, characterized in that: The viable count of Lactobacillus plantarum WL05 in step S4 is not less than 2×10 12 CFU / mL.

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