CN114921309A

CN114921309A - Asparagus liver-protecting wine and preparation and application thereof

Info

Publication number: CN114921309A
Application number: CN202210368384.7A
Authority: CN
Inventors: 周爱梅; 谭艳
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-04-08
Filing date: 2022-04-08
Publication date: 2022-08-19

Abstract

The invention provides asparagus liver-protecting wine and preparation and application thereof. The invention prepares asparagus fermented wine by scientific fermentation of asparagus, firstly, the asparagus is added with water and then crushed, the pH value is adjusted, composite pectinase is added for enzymolysis, the filtration and the sterilization are carried out, the sugar degree is adjusted, saccharomyces cerevisiae is inoculated for main fermentation, the filtration and the after-fermentation are carried out, and the asparagus fermented wine is obtained after clarification. According to the invention, the validamia glauca is selected as a raw material, a fermentation system is scientifically designed according to the characteristics of the validamia glauca, the raw material treatment mode, enzymolysis, fermentation, clarification and other processes of the validamia glauca are researched in a targeted manner, the fermentation process is optimized integrally, active substances in the asparagus raw material are well reserved, the content of liver protection active substances such as total phenolic acid and the like is obviously improved, and the validamia glauca has excellent liver protection activity. On the basis, the asparagus liver-protecting wine is prepared by selecting extracts of kudzuvine root, medlar, schisandra chinensis and hovenia dulcis thunb and compounding, the compatibility is scientific, and the obtained asparagus liver-protecting wine has better liver-protecting activity, good taste and high comprehensive nutritional value.

Description

Asparagus liver-protecting wine and preparation and application thereof

Technical Field

The invention belongs to the technical field of fermented wine preparation. More particularly relates to asparagus liver-protecting wine and preparation and application thereof.

Background

Asparagus (Asparagus officinalis L), also known as Asparagus officinalis, Asparagus and Asparagus, is a rare vegetable used as both medicine and food, called "vegetable king", and is also the first of ten healthy vegetables recognized by the world health organization. The medicinal value of asparagus is listed as 'above the top grade' in the compendium of materia medica, the position is only second to ginseng, and the asparagus has the effects of tonifying qi, reducing phlegm, refreshing the stomach and the like according to records. The asparagus is rich in 18 amino acids and various trace elements such as calcium, iron, phosphorus, zinc, potassium, selenium and the like required by a human body, also contains various active ingredients such as flavone, saponin, phenolic acid, polysaccharide and the like, can effectively reduce blood pressure and blood fat and inhibit and soften angiopathy after being eaten frequently, and also has various physiological functions such as cancer prevention, cancer resistance, liver protection, aging resistance, blood fat reduction and the like.

But as a medicine and food dual-purpose material, the development of health care and medicinal effects has a great relationship with the promotion and enrichment of active ingredients thereof. The prior art discloses a brewing process of asparagus wine, which is characterized in that asparagus and grain raw materials (sticky rice, broom corn millet, fragrant rice and oat) are fermented and brewed together, but the asparagus wine obtained by the method only can keep the original nutrient substances in the asparagus, the content of active ingredients is reduced, and the health-care and medicinal effects are not ideal, so that the exploration of the technical means for improving the content of the active substances in the asparagus has important significance.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the preparation method of the fermented wine capable of remarkably increasing the content of the active substances of the asparagus, so that the liver protection activity of the fermented wine is enhanced, and the fermented wine has good taste and high comprehensive nutritional value.

The second purpose of the invention is to provide the asparagus fermented wine prepared by the method.

The third purpose of the invention is to provide a preparation method of asparagus liver-protecting wine.

The fourth purpose of the invention is to provide the asparagus liver-protecting wine prepared by the method.

The fifth purpose of the invention is to provide the application of the asparagus liver-protecting wine in preparing liver-protecting products.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a preparation method of asparagus fermented wine, which comprises the following steps:

s1, adding water into asparagus, crushing, adjusting pH, adding compound pectinase for enzymolysis, and filtering;

s2, sterilizing, and adjusting the sugar degree to be 24-30 degrees Brix;

s3, adding saccharomyces cerevisiae, performing main fermentation for 5-7 days at the temperature of 24-27 ℃ and the pH value of 3.2-3.8, filtering, and performing after-fermentation;

and S4, adding gelatin and bentonite for clarification to obtain the asparagus fermented wine.

Preferably, the asparagus of S1 is validamia glauca.

Aiming at the characteristics of the valilla asparagus, the invention carries out targeted exploration on the processes of the valilla asparagus such as raw material processing mode, enzymolysis, fermentation, clarification and the like through continuous creative tests, so that the contents of total phenolic acid and the like of the asparagus fermented wine are obviously improved compared with the asparagus raw material, and the asparagus fermented wine has excellent liver protection activity.

Preferably, the asparagus of S1 is also pretreated before being added with water, specifically, washed clean and then drained.

Preferably, the mass ratio of the asparagus to the water of S1 is 1:1.5 to 2.

Preferably, the pH of S1 is 2-4, and most preferably 3.

Preferably, the final concentration of the composite pectinase S1 is 0.2-0.4%, and most preferably 0.3%.

Preferably, the composite pectinase of S1 comprises pectinase and cellulase.

Preferably, the enzymolysis of S1 is carried out at 45-55 ℃ for 1.5-2.5 h, and most preferably at 51 ℃ for 2 h.

Preferably, the filtering of S1 is 120 mesh filtering.

Further preferably, the filtering is performed by using two 120-mesh gauze layers.

Preferably, the sterilization of S2 is sterilization using a bactericide comprising sulfur dioxide and/or potassium metabisulfite.

Further preferably, the final concentration of the sulfur dioxide and/or the potassium metabisulfite is 110-130 mg/L, and most preferably 120 mg/L.

Preferably, after the sterilization in the S2, the product is still kept stand for 10-14 hours, so that solid matters are precipitated, the sterilization agent can fully exert the function of the product, and the activity of the Saccharomyces cerevisiae in the S3 cannot be influenced.

Preferably, citric acid is further added to adjust the pH to 3-4, most preferably 3.5 after the sterilization in S2.

Preferably, the sugar degree adjustment in S2 is performed using sucrose or crystal sugar.

Preferably, the sugar degree of S2 is 27 ° Brix.

Preferably, the saccharomyces cerevisiae of S3 needs to be activated before being inoculated.

Further preferably, the method of activation is: activating the mixture for 20 to 30min in distilled water with the sugar content of 4 to 6 percent at the temperature of 35 to 40 ℃.

Preferably, the S3 saccharomyces cerevisiae is l.a.13 yeast.

Preferably, the final concentration of the Saccharomyces cerevisiae S3 is 0.05-0.15% (w/v), and most preferably 0.067% (w/v).

Preferably, the fermentation is carried out at 24 ℃ and pH 3.5 for 6 days after the inoculation of S3 with saccharomyces cerevisiae.

Preferably, the post-fermentation of S3 is carried out at 13-17 ℃ for 23-27 days. Most preferably at 15 ℃ for 25 days.

The fermentation in the method is divided into two times, the first time is main fermentation, the sugar in the fermentation liquor is rapidly degraded by the saccharomyces cerevisiae to generate alcohol, when the alcohol concentration reaches a certain amount (after 5-7 days of fermentation), after-fermentation is carried out after filtration, the fermentation can be carried out in another clean fermentation tank, and the purpose of the after-fermentation is to slowly degrade the residual sugar in the fermentation liquor by the saccharomyces cerevisiae, and the aroma and the color of the wine are more coordinated.

Preferably, the mass ratio of the gelatin to the bentonite in S4 is 1: 2.7-3.2, most preferably 1:3.

further preferably, the total mass of the gelatin and the bentonite is 0.3-0.5% of the volume of the asparagus fermented wine, and most preferably 0.4%.

Preferably, the clarification is carried out at 20-30 ℃ for 32-38 h, and most preferably at 25 ℃ for 35 h.

Aiming at the characteristics of the validamia odorata, the invention researches the raw material processing mode, enzymolysis, fermentation, clarification and other processes of the validamia odorata through continuous creative tests, and simultaneously closely tracks the content of each component and the dynamic change of flavor substances in each link, so that the content of total phenolic acid and the like of the asparagus fermented wine is obviously improved compared with the asparagus raw material, and the asparagus fermented wine has excellent liver protection activity, therefore, the asparagus fermented wine prepared by the method is within the protection range of the invention.

The invention also provides a preparation method of the asparagus liver-protecting wine, which comprises the following steps:

s1, pressing the schisandra chinensis, the medlar, the radix puerariae and the hovenia dulcis thunb according to the ratio of 3-5: 4-6: 4-6: 4-6, sequentially crushing, ultrasonically extracting, concentrating and drying to obtain a traditional Chinese medicine dry paste;

s2, adding the traditional Chinese medicine dry paste obtained in the step S1 into the asparagus fermented wine in the claim 6, uniformly mixing, filtering and sterilizing to obtain the asparagus liver-protecting wine.

Preferably, the mass ratio of the total mass of the schisandra chinensis, the medlar, the kudzuvine root and the hovenia dulcis thunb to the mass of the water in the step S1 is 1: 10-12.5, most preferably 1: 12.5.

preferably, the crushed S1 is further kept stand for 10-14 hours, and most preferably 12 hours, so that active ingredients in the traditional Chinese medicine raw materials can be better dissolved out.

Preferably, the ultrasonic extraction of S1 is ultrasonic extraction at 42-48 ℃ for 1.5-2.5 h, and most preferably ultrasonic extraction at 45 ℃ for 2 h.

Further preferably, the number of times of ultrasonic extraction is 4-5 times, and most preferably 4 times.

More preferably, the residue is discarded after the ultrasonic extraction, and the filtrates are combined.

Preferably, the concentration of S1 is concentration with a rotary evaporator.

Preferably, the drying of S1 is drying with a freeze dryer.

Preferably, the dosage ratio of the schisandra chinensis, the medlar, the kudzu root, the hovenia dulcis thunb and the asparagus fermented wine is 3-5 g: 4-6 g: 4-6 g: 4-6 g: 100 mL. Most preferably 4 g: 5 g: 5 g: 5 g: 100 mL.

Preferably, the S2 is still kept for 22-26 hours after being uniformly mixed, and the most preferred time is 24 hours.

On the basis of the asparagus fermented wine, the asparagus fermented wine is compounded with extracts of kudzuvine root, medlar, Chinese magnoliavine fruit and hovenia dulcis thunb in a specific proportion, the compatibility is scientific, the nutritional ingredients of asparagus are fully extracted and utilized, the obtained asparagus liver-protecting wine has good taste and high nutritional value, can effectively improve alcohol-induced hepatocyte degeneration, can improve the GSH content in mouse liver tissues, reduce the MDA content, improve the anti-oxidative stress capability of livers, improve the TG content in the liver tissues, regulate lipid metabolism, achieve more excellent liver-protecting effect, and meet the evaluation standard of health-care products in a handbook of 2020 edition health-care food function inspection and evaluation methods. Therefore, the asparagus liver-protecting wine prepared by the method and the application of the asparagus fermented wine or the asparagus liver-protecting wine in preparing liver-protecting products are all within the protection scope of the application.

The invention has the following beneficial effects:

The asparagus liver protection wine is also compounded with the extracts of the kudzuvine root, the medlar, the schisandra chinensis and the hovenia dulcis thunb in a specific proportion, so that the nutrient components of the asparagus are fully extracted and utilized, and the obtained asparagus liver protection wine has good taste and high nutritive value, can effectively improve the alcohol-induced hepatocyte degeneration, improves the GSH and TG content in the liver tissue of a mouse, reduces the MDA content and realizes more excellent liver protection activity.

Drawings

FIG. 1A shows the results of measuring the juice yield and the soluble solid content in different juice ratios, and FIG. 1B shows the results of measuring the total phenolic acid, total flavone and total sugar in different juice ratios.

Fig. 2A is a result of measuring the juice yield and the content of soluble solids after three enzymes are subjected to enzymolysis, fig. 2B is a result of measuring the content of total phenolic acids after three enzymes are subjected to enzymolysis, fig. 2C is a result of measuring the content of total flavonoids after three enzymes are subjected to enzymolysis, and fig. 2D is a result of measuring the content of total sugars after three enzymes are subjected to enzymolysis.

Fig. 3A is a result of measuring the juice yield and the content of soluble solids at different enzymolysis temperatures, and fig. 3B is a result of measuring the content of total phenolic acids, total flavonoids and total sugars at different enzymolysis temperatures.

Fig. 4A is a result of measuring the juice yield and the content of soluble solids at different enzymolysis times, and fig. 4B is a result of measuring the content of total phenolic acids, total flavonoids and total sugars at different enzymolysis times.

Fig. 5A is a result of measuring the juice yield and the content of soluble solids under different enzymatic hydrolysis pH, and fig. 5B is a result of measuring the content of total phenolic acids, total flavonoids, and total sugars under different enzymatic hydrolysis pH.

FIG. 6 shows the results of the measurement of the indexes of fermented asparagus wine in different yeasts.

FIG. 7 shows the results of the index measurement of the asparagus fermented wine with different amounts of potassium metabisulfite added.

FIG. 8 shows the results of the measurement of the indexes of fermented asparagus wine at different initial sugar levels.

FIG. 9 shows the results of the index measurement of asparagus fermented wine with different amounts of yeast.

FIG. 10 shows the results of the measurement of the indexes of fermented asparagus wine at different initial pH values.

FIG. 11 shows the results of the measurement of the indexes of fermented asparagus wine at different fermentation temperatures.

FIG. 12 shows the results of the index measurement of fermented asparagus wines at different fermentation times.

FIG. 13 is a graph showing the results of the contents of total flavonoids and total phenolic acids in asparagus fermented wine.

FIG. 14A is a spatial distribution diagram of samples of volatile components in fermented asparagus wine, and FIG. 14B is a graph of the component load of volatile components in fermented asparagus wine.

FIG. 15 shows the results of measurement of physicochemical components during fermentation.

FIG. 16 shows the results of measurement of the change of total effective components during fermentation.

Figure 17 is a radar plot of electronic nose sensor response intensity during fermentation.

FIG. 18A is a photograph of fermented wine of asparagus, and FIG. 18B is a photograph of liver-protecting wine of asparagus.

FIG. 19 is the results of HE staining of mouse liver.

FIG. 20 is the results of GSH content in mouse liver.

FIG. 21 shows the results of MDA content in mouse liver.

FIG. 22 shows the results of TG content in mouse liver.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

One, material source

Asparagus, valilla asparagus, ji an valia agro biotechnology limited; composite pectinase, cellulase and pectinase, food grade, Henan Shuojiong industry Co., Ltd; angel yeast, food grade, Angel yeast, Inc.; uvaferm CM yeast, L.A.13 yeast, Lalvin 71B yeast, food grade, LAFFORT, France; gelatin, chitosan, food grade, henan Xin and Biotech limited; bentonite, diatomaceous earth, food grade, henna, biotechnology limited; PVPP, food grade, Nicoti Diboshi self brewing machine Co.

Second, the measuring method

1. Determination of functional Components

The total polysaccharide is measured by a phenol-sulfuric acid method; the content of total phenolic acid is measured by Folin-phenol method; the content of total flavonoids is determined by a sodium nitrite-aluminum nitrate-sodium hydroxide color development method.

2. HPLC determination of monomer content of phenolic acid compound

Chromatographic conditions are as follows: column Eclipse Plus C18 (250X 4.6mm, 5 μm, Agilent); 1% acetic acid aqueous solution (a), methanol (B); elution conditions: 0-10 min, 5% (B); 10-42 min, 5% → 40% (B); 42-50 min, 40% → 65% (B); 50-60 min, 65% (B); 60-70 min, 65% → 5% (B); sample introduction amount: 10 mu L of the solution; flow rate: 1 mL/min; column temperature: 30 ℃; detection wavelength: 320 nm.

3. HPLC determination of flavonoid monomer content

The detection conditions are as follows: column Eclipse Plus C18 (250X 4.6mm, 5 μm, Agilent); 1% aqueous acetic acid (a), methanol (B); elution conditions: 0-15 min, 5% (B); 15-55 min, 5% → 50% (B); 55-75 min, 50% → 70% (B); 75-80 min, 70% → 80% (B); 80-85 min, 80% (B); 85-95 min, 80% → 5% (B); sample introduction amount: 10 mu L of the solution; flow rate: 1 mL/min; column temperature: 30 ℃; detection wavelength: 280 nm.

Example 1 enzymolysis juicing process test of asparagus fermented wine

Index measuring method

(1) Determination of juice yield

Cleaning Germinatus Phragmitis, cutting into small pieces, weighing, and recording weight m ₁ Weighing and recording the weight m of the asparagus juice after juicing ₂ And calculated according to the following formula:

(2) determination of soluble solid content

Measured using a handheld glucometer.

Secondly, determining the juice ratio

Taking 50g of asparagus, adding water according to the material-liquid ratio (w/w) of 1:0, 1:1, 1:1.5, 1:2, 1:2.5, 1:3 and 1:3.5 respectively, crushing to obtain asparagus juice, passing the asparagus juice through two layers of 120-mesh gauze, measuring the juice yield and the content of soluble solid matters, centrifuging at 8000r/min for 20min, and measuring the content of total phenolic acid, total flavone and total sugar.

The results are shown in FIG. 1, in which FIG. 1A shows the results of measuring the juice yield and the soluble solid content in different juice ratios, and FIG. 1B shows the results of measuring the total phenolic acid, total flavone and total sugar in different juice ratios.

It can be seen that, as the feed-liquid ratio increases, the content of functional components such as total flavonoids, total phenolic acids and total sugars in the asparagus juice also shows a descending trend, the content of soluble solids also shows a descending trend, and decreases from 5.5% to 0.4%, and the juice yield shows an ascending trend, and increases from 38.46% to 74.67%, which indicates that the concentration of the nutritional components in the asparagus juice is diluted with the increase of the water addition amount, but the juice yield can be increased. As can be seen from FIG. 3.3, the juice yield does not increase significantly (p >0.05) after the feed/liquid ratio is greater than 1:2(w/w), so the feed/liquid ratio (w/w) in the juicing operation of the present invention is most preferably 1:2.

Thirdly, single factor test of enzymolysis process

(1) Determination of enzyme type and amount added (final concentration)

Weighing 50g of asparagus, adding water until the material-liquid ratio (w/w) is 1:2, adjusting the pH value to 3, crushing and juicing, respectively adding cellulase, pectinase and composite pectinase until the final concentrations of the enzymes are 0.1%, 0.2%, 0.3%, 0.4% and 0.5% (w/v), performing enzymolysis for 2h in a water bath at 50 ℃, passing through two layers of 120-mesh gauze, determining the juice yield and the content of soluble solids, centrifuging at 8000r/min for 20min, and determining the content of total phenolic acid, total flavone and total sugar.

The results are shown in fig. 2, in which fig. 2A is the results of measuring the juice yield and the soluble solid content after three enzymes are subjected to enzymolysis, fig. 2B is the results of measuring the total phenolic acid content after three enzymes are subjected to enzymolysis, fig. 2C is the results of measuring the total flavone content after three enzymes are subjected to enzymolysis, and fig. 2D is the results of measuring the total sugar content after three enzymes are subjected to enzymolysis.

As can be seen from fig. 2, when the enzyme addition amount is less than 0.5%, the three enzymes have no significant difference (p >0.05) in the soluble solid content, total flavonoids and total sugar content in the asparagus juice under the same addition amount, and when the compound pectinase is selected for enzymolysis, the total phenolic acid content in the asparagus juice is significantly higher than that of the other two enzymes (p <0.05), and when the addition amount is greater than 0.2%, the juice yield of the asparagus under the action of the compound pectinase is also significantly higher than that of the other two enzymes (p <0.05), which indicates that the use of the compound pectinase for enzymolysis is more beneficial to the exudation of nutrient components and the increase of the juice yield in the asparagus juicing process, so the enzyme used in the enzymolysis process of the invention is the compound pectinase.

In addition, as can be seen from fig. 2, the addition amount of the composite pectinase has no significant influence on the soluble solid content and the total sugar content (p > 0.05); the juice yield, the total phenolic acid content and the total flavone content reach the maximum value when the addition amount is 0.3 percent. Therefore, the enzyme addition amount (final concentration) in the enzymatic hydrolysis process of the present invention is most preferably 0.3% (w/v).

(2) Determination of the temperature of the enzymatic hydrolysis

Weighing 50g of asparagus, adding water until the material-liquid ratio (w/w) is 1:2, adjusting the pH value to 3, crushing and juicing, adding 0.3% of compound pectinase, respectively placing in constant-temperature water bath of 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 60 ℃ for enzymolysis for 2h, passing through two layers of 120-mesh gauze screens, measuring the juice yield and the content of soluble solids, centrifuging at 8000r/min for 20min, and measuring the content of total phenolic acid, total flavone and total sugar.

The results are shown in fig. 3, in which fig. 3A shows the results of measuring the juice yield and the content of soluble solids at different enzymolysis temperatures, and fig. 3B shows the results of measuring the content of total phenolic acids, total flavonoids and total sugars at different enzymolysis temperatures.

As shown in FIG. 3A, the yield of the asparagus juice increases and then decreases with the increase of the enzymolysis temperature, and reaches the highest value at 50 ℃. Therefore, the enzymolysis temperature in the enzymolysis process of the invention is most preferably 50 ℃.

(3) Selection of the time of the enzymatic hydrolysis

Weighing 50g of asparagus, adding water until the material-liquid ratio (w/w) is 1:2, adjusting the pH value to 3, crushing and juicing, adding 0.3% of compound pectinase, performing enzymolysis for 0.5h, 1h, 1.5h, 2h and 2.5h in a constant-temperature water bath at 50 ℃, passing through two 120-mesh gauze layers, measuring the juice yield and the content of soluble solid matters, centrifuging at 8000r/min for 20min, and measuring the content of total phenolic acid, total flavone and total sugar.

The results are shown in fig. 4, in which fig. 4A shows the results of measuring the juice yield and the soluble solid content at different enzymolysis times, and fig. 4B shows the results of measuring the contents of total phenolic acid, total flavone and total sugar at different enzymolysis times.

As can be seen from FIG. 4, different enzymolysis times have no significant influence on the content of soluble solids and the content of each functional component in the asparagus juice (p >0.05), and the juice yield increases and then decreases with the extension of the enzymolysis time and reaches the highest value when the enzymolysis is carried out for 2 hours. Therefore, the enzymolysis time in the enzymolysis process of the invention is most preferably 2 h.

(4) Selection of enzymatic pH

Weighing 50g of asparagus, adding water until the material-liquid ratio (w/w) is 1:2, respectively adjusting the pH value to 2, 3, 4, 5 and 6, crushing and juicing, adding 0.3% of compound pectinase, carrying out enzymolysis for 2h in a constant-temperature water bath at 50 ℃, passing through two 120-mesh gauze layers, measuring the juice yield and the content of soluble solids, centrifuging at 8000r/min for 20min, and measuring the content of total phenolic acid, total flavone and total sugar.

The results are shown in fig. 5, in which fig. 5A shows the results of measuring the juice yield and the soluble solid content at different enzymatic pH, and fig. 5B shows the results of measuring the total phenolic acid content, the total flavone content and the total sugar content at different enzymatic pH.

As can be seen from FIG. 5, different initial pH values have no significant effect on the soluble solid content and the content of each functional component of the asparagus juice (p >0.05), and the juice yield of the asparagus gradually increases with the increase of the pH value, but has no significant increase after the pH value is greater than 3(p > 0.05). Therefore, the enzymolysis pH in the enzymolysis process of the invention is most preferably 3.

Fourthly, establishing an enzymolysis response surface test-regression model and analyzing variance

On the basis of the single-factor test result, a four-factor three-level test is designed by taking the enzyme addition amount (A), the enzymolysis time (B), the enzymolysis temperature (C) and the enzymolysis pH (D) as investigation variables and taking the juice yield (Y) as a response value, and is shown in table 1. The enzymatic response surface experimental design and results are shown in table 2.

TABLE 1 level design of experimental factors for response surface of enzymatic hydrolysis process

Table 2 enzymolysis process response surface experimental design and results (n ═ 3)

And (3) performing multiple regression fitting on the response values of 29 test points in the table 2 by adopting Design-expert.V10.0.7 software to obtain a quadratic multiple regression equation of the juice yield and each factor as follows: the juice yield (%) was 89.94+0.31A-0.19B +1.02C +0.058D-0.27AB-0.35AC-0.13AD-0.25BC-1.19BD-0.66CD-1.57A ² -1.24B ² -1.77C ² -0.54D ² . As shown in the analysis of variance of the regression model in Table 3, the juice yield F of asparagus was 4.92, and the "p-value Prob F" model p was<0.01, the model factor has extremely significant meaning, and the distortion item P is 0.3507, so that no significant difference exists, the model is reliable, and the test result can be well presumed. As can also be seen from Table 3, the degree of conformity R of the asparagus juice yield model ² The fitting degree of the model is higher and the experimental error is small as 0.91 and is close to 1; corrected fitness of R ² _Adj 0.6623, the model is said to account for changes in 66.23% response values. The coefficient of variation CV is 1.03%, which reflects that the confidence of the model is high. Therefore, the regression equation model can be used to explain the design.

TABLE 3 analysis of variance of regression models

Note: the p value p-value Prob > F in the table, p <0.05, indicates that the factors of the model or the investigation have significant influence; p <0.01 for prob > F indicates that the influence of the model or the factors investigated is extremely significant.

Fifthly, verification of optimal enzymolysis process conditions

Through a regression model of Design-expert.V10.0.7 software, the optimal process conditions for systematically predicting the asparagus juice yield are as follows: the enzyme addition amount is 0.31%, the enzymolysis time is 1.95h, the enzymolysis temperature is 51.46 ℃, the enzymolysis pH is 2.9, and under the condition, the juice yield of asparagus predicted by a model can reach 90.11%. The enzyme addition amount is adjusted to be 0.3 percent according to the practical feasibility, the enzymolysis time is 2 hours, the enzymolysis temperature is 51 ℃, and the enzymolysis pH is 3.0. The verification is repeated for three times under the condition, the obtained asparagus juice yield is 90.72%, no significant difference (p is greater than 0.05) is found between the obtained asparagus juice yield and a predicted theoretical value, and the model is accurate and reliable and has good fitting performance within an error allowable range.

Example 2 fermentation Process test of Asparagus fermented wine

First, index measuring method

(1) Sugar degree measurement

The measurement was carried out by a hand-held glucometer.

(2) Acidity and alcohol determination

The method is executed by referring to the acidity and alcoholic strength measuring method in GB/T15038-.

Second, sensory evaluation method

In the embodiment, a sensory evaluation method of asparagus wine is formulated by referring to a sensory evaluation method of GB/T15037-2006 grape wine. An evaluation group consisting of 10 panelists evaluated samples of asparagus fermented wine in terms of color, aroma, taste and typicality according to the asparagus wine sensory evaluation table (Table 4).

TABLE 4 sensory evaluation table for asparagus wine

Third, strain activation

The saccharomycetes are activated in distilled water with 5 percent of sugar content at the temperature of 35-40 ℃ for 20-30 min and then used for subsequent tests.

Selection of yeast species

Adjusting sugar degree of enzyme-inactivated Germinatus Phragmitis juice to 24 ° Brix and pH to 3.5, adding 120mg/L K ₂ O ₅ S ₂ Standing for 12h, inoculating 0.1% Angel Saccharomyces cerevisiae, Uvaferm CM yeast, L.A.13 yeast, Lalvin 71B yeast, respectively, fermenting in a constant temperature incubator at 24 deg.C for 6d, and comparing alcoholic strength, total phenolic acid content, total flavone content and sensory evaluation score of 5 yeast fermented wine liquidsSelecting the most suitable strain for fermenting the asparagus fermented wine.

The results are shown in fig. 6, and of the five yeasts, the asparagus fermented wine fermented by the Angel yeast has lower alcoholic strength, sensory score, total flavone content and total phenolic acid content; the alcoholic strength of the asparagus fermented wine fermented by the Lalvin 71B yeast is obviously higher than that of other groups (p is less than 0.05), but the sensory score, the content of total flavonoids and total phenolic acid and the like are lower; the alcoholic strength, sensory score, total flavonoids and total phenolic acids of asparagus fermented wine fermented by Uvaferm CM yeast and raman yeast were improved compared to those of malvin 71B and angel yeast, but were lower than those of l.a.13 yeast. Thus, the yeast species in the fermentation process of the present invention is most preferably L.A.13 yeast.

Five, single factor test of main fermentation process

(1) Selection of the amount of potassium metabisulfite added

Adjusting sugar degree of enzyme-inactivated Germinatus Phragmitis juice to 24 ° Brix and pH to 3.5, respectively adding K of 80mg/L, 100mg/L, 120mg/L, 140mg/L, and 160mg/L ₂ O ₅ S ₂ Standing for 12h, inoculating 0.1% L.A.13 yeast, fermenting in 24 deg.C constant temperature incubator for 6d, measuring alcohol content, total phenolic acid content, total flavone content and sensory evaluation value of the wine liquid at the end of fermentation, and selecting optimum K ₂ O ₅ S ₂ The addition amount is as follows.

The results are shown in FIG. 7, where it can be seen that the K's are different ₂ O ₅ S ₂ The alcohol content of the asparagus fermented wine added with the alcohol is 8.8-10.77% vol, and when the added amount is 120mg/L, the alcohol content of the asparagus fermented wine is 10.77% vol, which is obviously higher than that of other groups (p)<0.05). Sensory score with K ₂ O ₅ S ₂ The addition amount is increased and then decreased, and the change trend of the addition amount is the same as that of the alcoholic strength. The content of total phenolic acid and total flavone is determined by K ₂ O ₅ S ₂ The addition amount increases and then decreases, and the total flavone content reaches the highest when the alcoholic strength is the highest. Therefore, the addition amount of the potassium metabisulfite in the fermentation process is most preferably 120 mg/L.

(2) Selection of initial brix

Respectively adjusting the enzyme-deactivated reedThe bamboo shoot juice has sugar degree of 18 ° Brix, 21 ° Brix, 24 ° Brix, 27 ° Brix, 30 ° Brix, pH of 3.5, and added K of 120mg/L ₂ O ₅ S ₂ Standing for 12h, inoculating 0.1% L.A.13 yeast, fermenting in 24 deg.C constant temperature incubator for 6d, measuring alcohol content, total phenolic acid content, total flavone content and sensory evaluation value of the wine liquid at the end of fermentation, and selecting optimum initial sugar degree.

As shown in fig. 8, it can be seen that the alcoholic strength, sensory score and total flavonoids were increased and then decreased with the increase of the initial sugar degree in the fermentation broth, and the total phenolic acid content was slightly increased. When the initial sugar degree is 27 degrees Brix, the alcoholic strength of the asparagus fermented wine is the highest, the sensory score and the total flavone content are obviously higher than other groups (p <0.05), and when the initial sugar degree exceeds 27 degrees Brix, the alcoholic strength shows a descending trend. Thus, the initial Brix in the fermentation process of the present invention is most preferably 27 ° Brix.

(3) Selection of the amount of Yeast added

Adjusting sugar degree of enzyme-inactivated Germinatus Phragmitis juice to 24 ° Brix and pH to 3.5, adding 120mg/L K ₂ O ₅ S ₂ Standing for 12h, inoculating 0.03%, 0.05%, 0.1%, 0.15%, 0.2% L.A.13 yeast, fermenting in 24 deg.C constant temperature incubator for 6d, measuring alcohol content, total phenolic acid content, total flavone content and sensory evaluation value of the wine liquid at the end of fermentation, and selecting the optimum yeast addition amount.

As shown in fig. 9, it can be seen that the alcohol content, sensory score and total phenolic acid content of the asparagus fermented wine tend to increase and decrease with the increase of the yeast addition, and the total flavonoids tend to increase. When the addition amount of the yeast is 0.04-0.1%, the alcoholic strength gradually increases along with the increase of the addition amount of the yeast, and when the addition amount is 0.1-0.2%, the alcoholic strength tends to decrease along with the increase of the addition amount of the yeast, which may be caused by early ending of fermentation due to the fact that sugar in raw materials is consumed by mass propagation of the yeast, so that the alcoholic strength is reduced, and the flavor of the product is further influenced. Therefore, the sensory score also tends to decrease when the amount of the compound is 0.1 to 0.2%. Therefore, the yeast is most preferably added in an amount of 0.1% in the fermentation process of the present invention.

(4) Selection of initial pH of fermentation

Adjusting sugar degree of enzyme-inactivated Germinatus Phragmitis juice to 24 ° Brix, pH to 2.8, 3.2, 3.5, 3.8, and 4.1, respectively, adding 120mg/L K ₂ O ₅ S ₂ Standing for 12h, inoculating 0.1% L.A.13 yeast, fermenting in a constant temperature incubator at 24 deg.C for 6d, measuring alcohol content, total phenolic acid content, total flavone content and sensory evaluation value of the wine liquid at the end of fermentation, and selecting optimum initial pH value.

As shown in fig. 10, it can be seen that the effect of the increase of initial pH of fermentation on the total flavone content and sensory score is increased and then decreased, and at pH 3.5, the sensory score of asparagus fermented wine is the highest and is significantly higher than that of other groups (p < 0.05); the alcoholic strength is in an increasing trend along with the increase of the initial pH value of the fermentation, and the content of the total phenolic acid is in a decreasing trend opposite to the change of the alcoholic strength. Therefore, the initial pH of fermentation in the fermentation process of the present invention is most preferably 3.5.

(5) Selection of fermentation temperature

Adjusting sugar degree of enzyme-inactivated Germinatus Phragmitis juice to 24 ° Brix and pH to 3.5, adding 120mg/L K ₂ O ₅ S ₂ Standing for 12 hr, inoculating 0.1% L.A.13 yeast, fermenting in 18 deg.C, 21 deg.C, 24 deg.C, 27 deg.C, and 30 deg.C constant temperature incubator for 6d, measuring alcohol content, total phenolic acid content, total flavone content and sensory evaluation value of the wine liquid at the end of fermentation, and selecting optimum fermentation temperature.

The results are shown in fig. 11, and it can be seen that the sensory score is firstly slowly increased and then decreased along with the increase of the fermentation temperature, and is highest at 24 ℃, and the sensory score is higher than 27 ℃ and 30 ℃ at 18 ℃ and 22 ℃, which indicates that the sensory quality of the asparagus fermented wine is easily reduced due to the overhigh fermentation temperature; the total sugar content of the asparagus fermented wine fermented at different temperatures is 10.3-14.6 g/L, the alcoholic strength is 10.57-11.97% vol, the alcoholic strength is in a trend of slowly rising and then falling along with the rising of the temperature, and the alcoholic strength is highest at 24 ℃, because the aging process of yeast is accelerated due to the overhigh temperature, so that the alcoholic production capacity of the asparagus fermented wine is influenced. Therefore, the fermentation temperature in the fermentation process of the present invention is most preferably 24 ℃.

(6) Selection of fermentation time

Adjusting sugar degree of enzyme-inactivated Germinatus Phragmitis juice to 24 ° Brix and pH to 3.5, adding 120mg/L K ₂ O ₅ S ₂ Standing for 12h, inoculating 0.1% L.A.13 yeast, fermenting in 24 deg.C constant temperature incubator for 4d, 5d, 6d, 7d, and 8d, measuring alcohol content, total phenolic acid content, total flavone content and sensory evaluation value of the wine liquid at the end of fermentation, and selecting the most suitable fermentation time.

As shown in fig. 12, it can be seen that the alcoholic strength is increased continuously with the increase of the fermentation time, the sensory score and the content of total flavonoids and total phenolic acids tend to increase first and then decrease, and the alcoholic strength is increased significantly before 6d of fermentation and then gradually decreases, because the yeast is a facultative strain, and aerobic propagation is performed in the early stage of fermentation, a large amount of oxygen and sugar are consumed, and the propagation of yeast is inhibited as the fermentation proceeds, the alcoholic fermentation is slowed, the alcohol yield is gradually decreased, and thus the alcoholic strength is not increased any more until reaching saturation, and therefore, the fermentation time in the fermentation process of the present invention is most preferably 6 d.

Sixth, response surface test of main fermentation process

On the basis of a single-factor test result, selecting initial sugar degree (A), initial pH (B), yeast adding amount (C) and fermentation time (D) as investigation variables, and carrying out a response surface test by taking total phenolic acid content and sensory evaluation as response values according to the design principle of Box-Behnken. Table 5 is a table of response surface test factor levels. Table 6 shows the experimental design and results of the response surface of the main fermentation process.

TABLE 5 fermentation Process response surface Experimental factor level design

TABLE 6 Main fermentation Process response surface Experimental design and results (n ═ 3)

Seventhly, influence of main fermentation process conditions on total phenolic acid content

(1) Regression model building and analysis of variance

After the multiple regression fitting is carried out on the total phenolic acid content in the table 6 through Design-expert.V10.0.7 software, a regression model equation taking the total phenolic acid content as an objective function is obtained as follows: y is ₁ The total phenolic acid content (mg/L) is 213.87+0.7A +0.45B +1.36C-4.72D-2.57AB-2.29AC-0.58AD +3.11BC +0.74BD +0.47CD-3.68A ² -8.36B ² -5.09C ² -3.83D ² . As can be seen from Table 7, fitting model equation Y ₁ P of (a)<0.05, indicating that the model is significant; the mismatching term is not significant at the 0.05 level (P is 0.395 & gt 0.05), which shows that the total phenolic acid content can be well predicted by fitting the model, and the fitted model equation can be used. Correlation coefficient R of fitting model ² 0.8781, indicating that the model fits well. In addition, the coefficient of variation of the model is 1.50%, and the experimental result is reliable within an acceptable range.

TABLE 7 analysis of variance

Note: in the table, the P value P-value Prob > F P <0.05 indicates that the factors of the model or the investigation have significant influence; p <0.01 for prob > F indicates that the influence of the model or investigational factors is extremely significant.

(2) Verification of fermentation optimization process parameters by taking total phenolic acid content as index

Analysis by software Design-expert.V10.0.7 gave: the optimal fermentation parameters taking the total phenolic acid content as a response value are as follows: the fermentation time was 5.4d, the yeast addition amount was 0.065%, the initial sugar degree was 27.36 ° Brix, and the initial fermentation pH was 3.50. The total phenolic acid content of the asparagus fermented wine obtained under the condition is 215.38 mg/L. In order to verify the effectiveness of the method, the method is adjusted according to the actual situation as follows: the fermentation time is 5d, the initial sugar degree is 27 degrees Brix, the yeast addition amount is 0.063 percent, the initial fermentation pH is 3.50, three parallel experiments are carried out under the condition, the total phenolic acid content of the asparagus fermented wine is 215.03mg/L, and the model is verified to be available within an error range.

Eighthly, influence of main fermentation process conditions on sensory score

(1) Regression model building and analysis of variance

After the multiple regression fitting is carried out on the sensory score result in the table 6 through Design-expert.V10.0.7 software, a regression model equation taking the sensory score as a target function is obtained as follows: y is ₂ Sensory score of 80.56-0.92A +0.44B +0.64C-2.46D +1.3AB +0.24AC-1.20AD +0.62BC + 7.5E ³ *BD-1.27CD-4.91A ² -4.87B ² -3.83C ² -2.46D ² . As can be seen from Table 8, fitting model equation Y ₂ P of (a)<0.05, which indicates that the model is significant; the mismatching item is not significant at the 0.05 level (P is 0.08 to 0.05), which shows that the model can well predict the sensory score of the asparagus fermented wine, and the fitted model can be used. Correlation coefficient R of fitting model ² 0.8732, the fitting degree of the model equation is good; in addition, the coefficient of variation of the model is 1.54%, which shows that the experimental result is reliable.

TABLE 8 analysis of variance of regression models

(2) Verification of fermentation optimization process parameters of asparagus fermented wine by taking sensory evaluation as index

The optimal fermentation parameters which are obtained by analyzing response surface software and take the sensory score as a response value are as follows: the fermentation time was 5.5d, the yeast addition was 0.067%, the initial sugar degree was 26.95 ° Brix, the initial pH was 3.51, and the sensory score of the asparagus fermented wine fermented under these conditions was 81.3. In order to verify the effectiveness of the method, the method is adjusted according to the actual situation as follows: the fermentation time is 6d, the addition amount is 0.067%, the initial sugar degree is 27 DEG Brix, the fermentation initial pH is 3.50, three parallel experiments are carried out under the fermentation condition, the sensory score of the asparagus fermented wine is 80.9, the relative error with the predicted value is 0.4%, and the model is further verified to be available.

Nine, selection of main fermentation process parameters

And (3) integrating the influences of the different fermentation conditions on the total phenolic acid content and the sensory score, and adjusting by combining with actual conditions to finally determine that the fermentation conditions are as follows: fermentation time 6d, addition amount 0.067%, initial sugar degree 27 ° Brix, initial pH 3.51. Under the condition, three parallel experiments are carried out, the total phenolic acid content of the obtained asparagus fermented wine is 214.45mg/L, the sensory score is 80.9, and the model equation is accurate within an error range.

Example 3 raw material treatment Process test of Asparagus in Asparagus fermented wine

Raw material processing method and sample collection

1. Selecting the validamia glauca which is not rotten and deteriorated to respectively carry out 4 treatments:

(1) treatment 1: the asparagus is crushed, and the asparagus juice is obtained after enzymolysis according to the optimal enzymolysis process obtained in the embodiment 1, and fermentation is carried out according to the optimal fermentation process obtained in the embodiment 2, namely, enzymolysis clear juice fermentation, which is hereinafter referred to as Z1.

(2) And (3) treatment 2: asparagus is crushed and filtered to obtain asparagus juice, and the asparagus juice is fermented according to the optimal fermentation process obtained in the embodiment 2, namely the clear juice is not subjected to enzymolysis for fermentation, and the fermentation process is hereinafter referred to as Z2.

(3) And (3) treatment: the asparagus is crushed, and is subjected to enzymolysis according to the optimal enzymolysis process obtained in the embodiment 1, and fermentation is carried out according to the optimal fermentation process obtained in the embodiment 2 without filtering, namely, the fermentation with dregs by enzymolysis, which is hereinafter referred to as Z3.

(4) And (4) treatment: after the asparagus is crushed, the asparagus is directly fermented according to the optimal fermentation process obtained in the embodiment 2, namely, the asparagus is not subjected to enzymolysis and is fermented with dregs, which is hereinafter referred to as Z4.

2. And (5) when the main fermentation of the asparagus fermented wine in different raw material treatment modes is finished, sucking the supernatant to be tested.

Second, method for measuring physicochemical components and sensory evaluation

The total acidity, alcohol content, total sugar, pH of the asparagus fermented wine were measured and subjected to sensory evaluation, and the results are shown in table 9.

TABLE 9 influence of different raw material treatment modes on physicochemical components and sensory score of fermented asparagus wine

Note: each data is mean ± SD (n ═ 3), with different letters representing significant differences (p < 0.05).

As can be seen from Table 9, different raw material treatment methods have small influence on the pH and the total acid of the asparagus fermented wine, wherein the pH of the asparagus fermented wine in four groups is 3.43-3.55, no significant difference exists, and the total acid content is 5.38-5.73 g/L, and the difference is small. The alcoholic strength and the total sugar are relatively greatly influenced by the raw material treatment mode, the alcoholic strength of the asparagus fermented wine in four groups is 6.2-11.9% vol, the alcoholic strength of Z1 is the highest and is remarkably higher than that of other three groups of samples (p <0.05), and the content of the total sugar in Z4 is the highest and is 19.2g/L and is remarkably higher than that of other three groups of samples (p < 0.05). In addition, from the perspective of whether the fermentation with dregs is carried out, the alcoholic strength of the Z3 and Z4 groups is respectively obviously lower than that of the Z1 and Z2 groups (p is less than 0.05), which indicates that the fermentation with dregs can not be beneficial to the production of alcoholic strength; from the perspective of enzymolysis, the alcohol content of the Z2 and Z4 groups is significantly lower than that of the Z1 and Z3 groups (p is less than 0.05), which indicates that the enzymolysis can promote the wine yield of the yeast to a certain extent. And the sensory score of the asparagus fermented wine obtained by clear juice fermentation is remarkably higher than that of residue-carrying fermentation (p is less than 0.05), the sensory score of the asparagus fermented wine obtained by enzymolysis fermentation is remarkably higher than that of non-enzymolysis fermentation (p is less than 0.05), and the sensory score of Z1 in the four groups of samples is the highest, so that the asparagus fermented wine obtained by enzymolysis clear juice fermentation has the best taste.

Third, non-volatile component comparative analysis

(1) Comparative analysis of total efficacy components

The total flavone content and the total phenolic acid content of the asparagus fermented wine are measured, and the results are shown in fig. 13, and it can be seen that the total flavone content of the asparagus fermented wine in four groups is as follows from high to low: z4, Z3, Z2, Z1, wherein the total flavone content in Z4 is significantly higher than in the other three groups of samples (p < 0.05); the total phenolic acid content in the four groups of samples is as follows: z1> Z3> Z2> Z4, and the total phenolic acid content in Z1 is significantly higher than that of the other three groups of samples (p < 0.05). Therefore, under the same conditions, the total flavone content in the asparagus fermented wine obtained by residue-carrying fermentation is higher than that of a sample obtained by clear juice fermentation, and the total phenolic acid content in the asparagus fermented wine obtained by enzymolysis fermentation is higher than that of a group obtained by non-enzymolysis fermentation, which indicates that the enzymolysis can promote the formation of polyphenols in the asparagus fermented wine to a certain extent, and the total flavone content in the wine can be improved by residue-carrying fermentation.

(2) Comparative analysis of monomer functional component content

The contents of the functional components of the monomers in the asparagus fermented wine obtained by fermenting different raw material treatment modes are shown in table 10, and the contents of the phenolic acid monomer compounds in the asparagus fermented wine are higher than those of the flavonoid monomer compounds. The content of each monomer compound in four groups of asparagus fermented wine samples is different, wherein the content of naringin, hesperidin, phlorizin, nicotiflorin and narcissus in the four groups of asparagus fermented wine samples is not obviously different (p is more than 0.05); the content of rutin, chlorogenic acid, caffeic acid and coumaric acid in Z4 is significantly higher than that of other three groups of samples (p < 0.05); quercetin and vanillic acid are present in relatively high amounts in Z1, Z2 and Z4, and in relatively low amounts in Z3; kaempferol is relatively high in Z1 and Z2, and low in Z3 and Z4; and the content of gallic acid in Z2 and Z4 is obviously higher than that in Z1 and Z3(p < 0.05). In conclusion, the total number of the monomeric compounds with higher content in Z4 is more than that in other three groups of samples, which is probably due to the relatively higher content and types of the functional components in the asparagus slag.

TABLE 10 influence of different raw material treatment methods on the content of functional components in asparagus fermented wine monomer

Note: data are presented as mean ± SD (n ═ 3), with different letters indicating significant differences (p < 0.05).

Analysis of volatile Components

1. Electronic nose analysis

The volatile components in the asparagus fermented wine are measured by adopting the electronic nose, and the result is shown in fig. 14, so that the volatile substance distribution condition of the asparagus fermented wine obtained by fermenting different raw material treatment modes is shown in the PCA spatial distribution diagram. The contribution rates of the two main components are 76.28% and 11.13%, respectively, and nearly 90% of the total variance is explained, which shows that the two main components can explain the main characteristic information of four groups of asparagus fermented wine samples. Four groups of asparagus fermented wine samples were increased on PC1 in the order of Z2< Z1< Z3< Z4, but not significantly ranked on PC 2. The spatial regions of the four sets of samples show that the Z1 and Z2, Z3 and Z4 samples are close to each other, which means that there is a large difference in volatile matter composition between the march and juice fermented samples. When correlating the sample spatial profile (fig. 17A) and the component loading profile (fig. 17B), it can be seen that the W1C (aromatic, benzene) and W5C (short chain alkanes and aromatic) sensors contributed the most to Z2, the W3C (aromatic sensitive, ammoniac) sensors contributed the most to Z1, the W1W (sulfide sensitive) sensors contributed the most to Z3, and the W5S (sensitive to nitrogen oxides), W6S (sensitive to hydrides), W1S (sensitive to methyl), W2W (sensitive to aromatic), W2S (sensitive to alcohols, aldehydes, ketones) and W3S (sensitive to long chain alkanes) contributed the most to Z4. It can be readily seen from the above analysis that most sensors contributed more to the march-fermented samples, i.e., Z3 and Z4, indicating that the types and amounts of volatile substances in the march-fermented samples may be higher, probably because the march will lose some of the volatile substances in the asparagus skin dregs of the asparagus fermented wine.

2. HS-SPME-GC-MS analysis

(1) Analytical method

a. Headspace solid phase microextraction conditions

Extracting volatile substances in asparagus fermented wine by adopting an HS-SPME (headspace solid phase microextraction) method, aging DVB/CAR/PDMS extraction heads before use according to instructions respectively, adding 10mL of wine sample into a 40mL headspace extraction bottle, adding an internal standard substance cyclohexanone and 2g of sodium chloride, balancing for 10min at 40 ℃, inserting the aged extraction heads above the liquid level of the extraction bottle, extracting for 50min, and carrying out GC (gas chromatography) analysis for 5 min.

GC-MS detection conditions

GC conditions were as follows: the chromatographic column is a DB-WAX capillary column (60m × 0.25mm × 0.25 μm); the temperature of a sample inlet is 250 ℃; the carrier gas is high-purity helium (purity 99.999%): the column flow rate is 1.0 mL/min; no shunt sampling; temperature rising procedure: firstly keeping the temperature at 45 ℃ for 3min, heating to 140 ℃ at the speed of 6 ℃/min, then heating to 160 ℃ at the speed of 3 ℃/min, and finally heating to 210 ℃ at the speed of 4 ℃/min, and keeping the temperature for 8 min.

MS conditions: an electron ionization source (EI); the ionization voltage is 70 eV; the ion source temperature is 230 ℃; the temperature of the four-level bar is 150 ℃; the temperature of the transmission line is 280 ℃; the mass spectrum scanning range is 35-600 Da in m/z.

(2) HS-SPME/GC-MS analysis results

In the four groups of samples, 35 volatile substances were identified, including 7 types of esters, alcohols, aldehydes, ketones, acids, terpenes and phenols, and the specific types and contents of the volatile substances are shown in table 11. 30, 25, 24 and 23 volatile species were identified in the Z1, Z2, Z3, Z4 samples, respectively. The common volatile substances detected in the four groups of asparagus fermented wine comprise 19 types, including 9 types of ester compounds (ethyl acetate, amyl acetate, ethyl caproate, hexyl acetate, heptyl acetate, ethyl decanoate, ethyl octanoate, phenethyl acetate and ethyl laurate), 6 types of alcohol compounds (ethanol, isoamyl alcohol, n-hexanol, n-heptanol, 1-octanol and DL-menthol), 3 types of acid compounds (n-hexanoic acid, caprylic acid and n-decanoic acid) and 1 type of terpene compounds (styrene).

Table 11 shows the content of volatile substances in the asparagus fermented wine samples fermented by different raw material processing methods, and it can be seen from table 11 that there are differences in the types and contents of volatile substances in the asparagus fermented wine samples fermented by different raw material processing methods, wherein the influence of the fermentation with dregs on the volatile substances in the asparagus fermented wine is greater than the enzymolysis.

TABLE 11 content of volatile substances in samples of fermented asparagus wine fermented by different raw material treatment methods

Example 4 test of composition Change during fermentation of Asparagus fermented wine

First, collecting a sample

Selecting validamia glauca linn without putrefaction, crushing, performing enzymolysis according to the optimal enzymolysis process obtained in the example 1, taking asparagus juice, performing fermentation according to the optimal fermentation process obtained in the example 2, and extracting supernatant liquid during fermentation to perform component determination.

Secondly, the change of physicochemical components during the fermentation

The results of the measurement of the physicochemical components during the fermentation period are shown in fig. 15, the alcoholic strength of the asparagus fermented wine is continuously increased during the fermentation period, wherein the increasing rate of the main fermentation stage (0-6 d) is higher, and the increasing rate of the asparagus fermented wine is continuously reduced during the later fermentation period. The sugar degree is opposite to the change trend of the alcoholic strength, and the sugar degree does not change significantly after the main fermentation is finished (p is more than 0.05). The pH value is obviously reduced (p is less than 0.05) when the fermentation is carried out for 0-2 d, then the pH value is stabilized between 3.2-3.5, the total acid is firstly increased and then becomes stable along with the extension of the fermentation time, and the change trends of the total acid and the total acid have certain opposite rules.

Third, non-volatile component change during fermentation

(1) Variation of total effective component

As shown in fig. 16, the total phenolic acid content increases and then levels during the fermentation of the asparagus fermented wine, wherein during the main fermentation for 0-4 d, the total phenolic acid content increases significantly (p <0.05), and during the fermentation for 5-6 d, the total phenolic acid content decreases slightly compared with that during the 4 th d, while during the post fermentation, the total phenolic acid content increases slightly and then decreases, but the content change is not significant (p > 0.05); during the period of 0-5 d of main fermentation, the content of the total flavonoids is firstly obviously increased and then reduced at the 2 nd d, when the main fermentation is finished, the content of the total flavonoids is gradually increased to a level equal to that before the fermentation, and in the later fermentation stage, the content of the total flavonoids is not obviously changed just at the beginning until the 17 th d of the fermentation, the content of the total flavonoids is obviously reduced (p is less than 0.05), and then the total flavonoids tend to change in a vertically stable fluctuation mode.

(2) Variation of functional component content of monomer

In the process of fermenting the asparagus fermented wine, the content changes of 13 main phenolic acids and flavonoid monomeric compounds are monitored, wherein the main phenolic acids and the flavonoid monomeric compounds comprise naringin, hesperidin, rutin, phlorizin, nicotiflorin, narcissus, quercetin, kaempferol, gallic acid, vanillic acid, chlorogenic acid, caffeic acid and coumaric acid. The results of the change in the content of each compound are shown in tables 12 and 13.

From tables 12 and 13, it can be seen that the flavonoid monomer compounds are less in content during the fermentation process of the asparagus fermented wine, wherein the highest content is rutin, and the second is fireworks glycoside, narcissin and hesperidin. Besides quercetin, the contents of other 7 flavonoid monomer compounds in the 8 flavonoid monomer compounds are in a trend of increasing first and then decreasing during fermentation, and the contents are highest basically when the fermentation lasts for 3-4 days. Wherein the content of hesperidin and naringin reaches the highest in 3d fermentation, and the content of hesperidin and naringin shows a change trend of stable fluctuation up and down in the post-fermentation period. The content of the quercetin changes little during the fermentation period and is almost in a stable state; the variation trend of rutin content is similar to that of hesperidin and naringin; and the content of phlorizin, fireworks glycoside, narcissus and kaempferol gradually increases when the fermentation lasts for 0-4 d, and then shows a descending trend until the content gradually becomes stable after the fermentation lasts for 22 d.

In the fermentation process of the asparagus fermented wine, the content of the phenolic acid monomer compound is higher than that of the flavonoid monomer compound, which has positive influence on the color and luster of the asparagus fermented wine. The content of gallic acid in the 5 phenolic acid monomer compounds in the fermentation process of the asparagus fermented wine is the highest, the content of the gallic acid is in an ascending trend in the early stage of fermentation, the change is possibly related to the generation of the gallic acid by hydrolyzing ester substances, the content of the gallic acid reaches the highest in the 4 th day of fermentation, and then the content of the gallic acid begins to decrease until the content fluctuation is 387.60-407.02 mg/L in the later stage of fermentation. The vanillic acid and the chlorogenic acid are phenolic acid monomeric compounds with the content second to that of gallic acid in the fermentation process of the asparagus fermented wine, are gradually decomposed by microorganisms during the main fermentation period, the contents of the vanillic acid and the chlorogenic acid are in a descending trend, the contents of the vanillic acid and the chlorogenic acid are in a fluctuation change during the post fermentation period, the vanillic acid and the chlorogenic acid belong to hydroxycinnamic acids, and the reduction of the contents of the vanillic acid and the chlorogenic acid is possibly related to the degradation of the acidified compounds caused by polyphenol oxidase. Caffeic acid is the lowest compound in 5 phenolic acid monomer compounds, and the content of the caffeic acid is not changed greatly in the fermentation process and basically stabilized between 0.1 and 0.2 mg/L. The coumaric acid content varies from up-down-up-down with increasing fermentation time, which may be associated with condensation hydrolysis between coumaric acid and esters. In conclusion, the change degrees of the content changes of other substances except the quercetin and the caffeic acid in the main fermentation process are all larger than those in the post-fermentation stage.

TABLE 12 variation of the content of each monomer functional component during the main fermentation period of asparagus fermented wine (n ═ 3)

TABLE 13 variation of the content of each monomer of functional ingredient during post-fermentation of asparagus fermented wine (n ═ 3)

Fourthly, volatile components change during the fermentation

(1) Electronic nose analysis based volatile components in fermentation period of asparagus fermented wine

From the radar chart of the response intensity of the electronic nose sensor in fig. 17, it can be seen that the sensors with large change of response intensity to the asparagus fermented wine sample during fermentation are W5S, W2W, W2S, W1W and W1S, which are sensitive to nitrogen oxides, aromatic components, alcohols, aldehydes, ketones, sulfides and methyl compounds, respectively, and indicate that the aromatic components, alcohols, aldehydes, ketones, sulfides and methyl compounds in the asparagus fermented wine have large change during fermentation. In the fermentation process, the response intensity of the W2W sensor is continuously enhanced along with the extension of the fermentation time, which shows that the content of aromatic components in the asparagus fermented wine gradually rises in the fermentation period; and the response intensity of the sensors W5S, W2S, W1W and W1S is firstly enhanced and then weakened along with the prolonging of the fermentation time, which shows that the content of nitrogen oxides, alcohols, aldehydes and ketones, sulfides and methyl compounds in the asparagus fermented wine is firstly increased and then reduced during the fermentation period.

(2) GC-MS-based analysis of volatile components during fermentation of asparagus fermented wine

The analysis of volatile substances during the fermentation of asparagus fermented wine was performed using HS-SPME/GC-MS and the results are shown in tables 14 and 15. GC-MS identified a total of 41 volatile species including 10 esters, 13 alcohols, 9 aldehydes, 4 ketones, 3 acids, 1 phenol and 1 terpene.

As can be seen from Table 14, 25 volatile substances were detected in the asparagus juice at the 0 th fermentation stage, and Table 15 shows that the types of volatile substances in the asparagus fermented wine are greatly different as the fermentation progresses, and 33 volatile substances were detected in the main fermentation stage (1-6 d) and 27 volatile substances were detected in the post-fermentation stage (12-32 d). Compared with asparagus juice, the types of esters and alcohols in the main fermentation stage and the after-fermentation stage are remarkably increased, the types of aldehydes and ketones are remarkably reduced, and the result is consistent with the detection result of an electronic nose.

The change trend of each ester compound is basically consistent during the fermentation, the content of each ester compound is increased and changed in the main fermentation stage, and the content of each ester compound is reduced in the post fermentation stage. Ethyl acetate, isoamyl acetate, ethyl caproate, ethyl caprylate and phenethyl acetate are ester compounds with higher content in the fermentation process of the asparagus fermented wine. The alcohol compounds are the substances with the highest content and the most variety in the fermentation process of the asparagus fermented wine, the contents of ethanol, isobutanol, isoamyl alcohol and heptanol are in a slow rising trend along with the fermentation, wherein the increase of the content of the ethanol is most obvious, the isoamyl alcohol is used as the second component, the fruit wine is mainly endowed with mature fruit fragrance and grass fragrance, and the contents of the 2-ethyl-hexanol, the sec-octanol, the 1-octene-3-ol, the 1-octanol, the DL-menthol and the beta-phenethyl alcohol are in a rising-first and falling-second changing trend, and reach the maximum content at the end of the main fermentation. The change of carbonyl compounds such as aldehydes and ketones in the fermentation process of the asparagus wine is mainly reflected in that the types and the content of the carbonyl compounds are obviously reduced along with the extension of the fermentation time. In addition, the types of the acid compounds are relatively less in the process of fermenting the asparagus wine, the types and the content of the acid compounds are not greatly changed in the process of fermenting the asparagus wine, the content of the styrene is in an ascending trend along with the prolonging of the fermentation time, and the content of the 2, 4-di-tert-butylphenol serving as the aroma component of the asparagus juice is not obviously changed in the process of fermenting the asparagus juice.

TABLE 14 content variation of volatile substances during fermentation of Asparagus fermented wine

TABLE 15 content variation of volatile substances during post-fermentation of asparagus fermented wine

In conclusion, the alcoholic strength of the asparagus fermented wine is continuously increased during fermentation, wherein the increasing speed of the main fermentation stage (0-6 d) is higher, the increasing speed is continuously reduced during the subsequent fermentation, the sugar degree is continuously reduced, after the main fermentation is finished, the sugar degree change is not significant (p is greater than 0.05), the pH value is stabilized between 3.2 and 3.5, the acidity and the total phenolic acid content show an increasing trend, and the total flavone content shows a fluctuation change of reduction-increase-reduction. The content of each monomer phenol and monomer flavonoid substance changes differently during the fermentation period, but the change trend is greater in the main fermentation stage than in the post-fermentation stage. Along with the fermentation, the types of esters and alcohols in the asparagus fermented wine are increased continuously, the types of aldehydes and ketones are reduced remarkably, the contents of acids and phenols are relatively stable in the fermentation process of the asparagus fermented wine, the total change is not large, and the content of styrene is changed in an ascending way along with the extension of the fermentation time.

Example 5 preparation of Asparagus liver-protecting wine

Preparation of asparagus fermented wine

S1, washing and draining the validamia glauca, and adding water until the ratio of material to liquid is 1:2, crushing, adjusting the pH value to 3, adding 0.3% of compound pectinase, performing enzymolysis for 2 hours at 51 ℃, and filtering by using two layers of 120-mesh gauze;

s2, sterilizing with 120mg/L potassium metabisulfite, standing for 12 hours, and adding sucrose until the sugar degree is 24-27 degrees Brix;

s3, activating, adding L.A.13 yeast, fermenting at 24 ℃ and pH 3.5 for 6 days, filtering, replacing another clean fermentation tank, and fermenting at 15 ℃ for 25 days;

s4, adding the materials in a mass ratio of 1:3 to a final concentration of 0.4% (w/v), and clarifying at 25 deg.C for 35h to obtain the fermented wine (photograph is shown in FIG. 18A).

Second, preparation of asparagus liver-protecting wine

S1, adding 4g of schisandra chinensis, 5g of medlar, 5g of radix puerariae and 5g of semen hoveniae into water until the material-to-liquid ratio (w/w) is 1: 12.5, sequentially crushing, standing for 12h, performing ultrasonic extraction at 45 deg.C for 2h (4 times), discarding residues, mixing filtrates, concentrating with rotary evaporator, and drying with freeze dryer to obtain Chinese medicinal dry extract;

s2, adding the traditional Chinese medicine dry paste obtained in the step S1 into 100mL of asparagus fermented wine, uniformly mixing, standing for 24h, filtering and sterilizing to obtain the asparagus liver-protecting wine (the picture is shown in figure 18B).

Example 6 in vivo hepatoprotective Activity study of Asparagus liver-protecting wine

First, experiment method

1. Grouping and administration modeling of experimental animals

84 male KM mice were randomly assigned to 7 groups after adaptive feeding for 7d as shown in table 16. The mice are filled with 1mL/100g of stomach, the blank group and the model group are filled with stomach distilled water, the negative group is filled with 11% edible alcohol, the positive group is filled with stomach silybin, and each sample group (high, medium and low dose groups of asparagus liver-protecting wine) is filled with stomach asparagus liver-protecting wine, and the stomach filling is continuously carried out for 30 days. After 30 days, the model group, the positive group, the negative group and each sample group were subjected to intragastric administration of 0.3mL/20g of 50% ethanol at a time, and the blank group was subjected to intragastric administration of distilled water, and the animals were sacrificed after fasting for 16 hours. After the blood sampling is finished, the abdominal cavity is cut open, and the liver is taken out for measuring each index.

TABLE 16 animal grouping and dosing schedules

2. Influence of asparagus liver-protecting wine on organ index and liver pathological changes of mice

(1) Pathological changes of liver

Fixing liver tissues in a 10% neutral formaldehyde solution, placing the fixed liver tissues in a tissue embedding box, washing with running water, and dehydrating with gradient ethanol from low to high; the tissue is soaked in xylene for 1 hour, so that the paraffin can be completely soaked in the tissue, and the process is transparent; after the tissues are subjected to transparent treatment, the tissues are soaked in paraffin for 4 hours, and the liver tissues after being soaked in the paraffin are placed in an embedding box and embedded by the paraffin. Cutting the wax block coated with the tissue into tissue sections with the thickness of 3um by using a paraffin slicer, and baking the glass slide with the tissue at the temperature of 60 ℃ for 1-2 h; finally, paraffin sections were stained with HE and the pathological morphology of the liver tissue was observed under an optical microscope.

(2) Detection of asparagus liver-protecting wine on various indexes of mouse liver tissues

Taking the supernatant of the liver tissue homogenate, and detecting the content of Glutathione (GSH), Malondialdehyde (MDA) and Triglyceride (TG) in the liver tissue of each group of mice.

Second, experimental results

1. Pathological section analysis of mouse liver

Results of HE staining of mouse liver are shown in fig. 19: the hepatocytes of the blank group of mice (fig. 19A) are regularly and radially arranged, the cell morphology is normal, the sizes are uniform, the cell boundaries are complete, and the cell nuclei are clearly visible; the liver lobe structures of the model group mice (fig. 19B and 19C) and the negative group mice (fig. 19D) are irregular, the cell morphology is unclear, the cell boundaries are fuzzy, and the arrangement is scattered; positive group mice (fig. 19E): the cell shape is normal, is close to a blank group, the size and the structure are uniform, the arrangement is neat, and the cell boundary is complete. Validus glauca hepatoprotective wine sample set (fig. 19F, fig. 19G, fig. 19H): the liver structure is basically recovered, the cell morphology is good, the liver cells of the low-dose group of the jinggangsha liver-protecting wine are closest to the blank group, the damage degree of the liver cells of the medium-dose group and the high-dose group is slightly high, but the liver cell morphology of each dose group of the jinggangsha liver-protecting wine sample is improved compared with that of the model group and the negative group, and the jinggangsha liver-protecting wine has a protection effect on the liver of a mouse with alcoholic liver damage.

2. Influence of asparagus liver-protecting wine on GSH content in liver

As shown in fig. 20, GSH levels in liver tissue of the model group mice were significantly reduced compared to the blank group (p < 0.01). Compared with a model group and a negative mouse, the GSH level of each dose group of the jinggangsha liver-protecting wine sample is increased, wherein the low dose group has a significant increase (p is less than 0.05), which shows that the low dose of the jinggangsha liver-protecting wine can significantly improve the GSH content in the liver tissue of the mouse.

3. Influence of asparagus liver-protecting wine on MDA content in liver

As shown in fig. 21, it can be seen that the MDA in liver tissues of the model group and the negative group was significantly increased (p <0.01) compared to the blank group, indicating successful modeling. Compared with the model group, the positive drug group has a significantly reduced MDA content (p <0.01), while the MDA content in the Jinggang asparagus liver protection wine sample group shows an upward trend with the increase of the dosage of the Jinggang asparagus liver protection wine, wherein the low dosage (p <0.01) and the medium dosage (p <0.05) have significant differences compared with the model group and the negative group.

4. Analysis of content of lipid metabolites in mouse liver

As shown in fig. 22, it can be seen that there is a very significant increase in TG content in both the model group and the negative group (p <0.01) compared to the blank group, while each dosage group of the asparagus liver-protecting wine sample has a significant effect of reducing TG content.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A preparation method of asparagus fermented wine is characterized by comprising the following steps:

s2, sterilizing, and adjusting the sugar degree to be 24-30 DEG Brix;

2. The method of claim 1, wherein the asparagus is valilla glauca.

3. The method of claim 1, wherein the adjusting the sugar degree in step S2 is performed by using sucrose or crystal sugar.

4. The method according to claim 1, wherein the post-fermentation in step S3 is carried out at 13-17 ℃ for 23-27 days.

5. The method according to claim 1, wherein the mass ratio of the gelatin to the bentonite in step S4 is 1: 2.7 to 3.2; the total mass of the gelatin and the bentonite is 0.3-0.5% of the volume of the asparagus fermented wine.

6. The asparagus fermented wine prepared by the method of any one of claims 1 to 5.

7. A preparation method of asparagus liver-protecting wine is characterized by comprising the following steps:

8. The method according to claim 7, wherein the dosage ratio of the schisandra chinensis, the medlar, the kudzuvine root, the hovenia dulcis thunb and the asparagus fermented wine is 3-5 g: 4-6 g: 4-6 g: 4-6 g: 100 mL.

9. The asparagus liver-protecting wine prepared by the method of claim 7 or 8.

10. Use of the asparagus fermented wine of claim 6 or the asparagus liver-protecting wine of claim 9 in the preparation of liver-protecting products.