CN107252093B - A kind of guava leaves rich in soluble polyphenols and flavonoid aglycones and preparation method and application - Google Patents

Abstract

The invention discloses a guava leaf rich in soluble polyphenols and flavonoid aglycones and a preparation method and application. In the present invention, the cleaned guava leaves are drained, dried, crushed and sieved to obtain the guava leaves; after mixing them with water, the pH value is adjusted, and enzymes are added to carry out enzymolysis reaction; The system after the enzymatic hydrolysis reaction is dried to obtain a guava leaf product rich in soluble polyphenols and flavonoid aglycones. The content of soluble polyphenols in the guava leaf tea obtained by the preparation method has been greatly improved, and the flavonoid glycoside components of guava leaves are degraded into flavonoid aglycone components with stronger functional activity, and the quercetin, kaempferol, etc. The aglycone content enhances the antioxidant and anti-DNA damage effects of guava leaves. Therefore, the guava leaves rich in soluble polyphenols and flavonoid aglycones have great application potential in the field of food and/or health care products.

Description

A kind of guava leaves rich in soluble polyphenols and flavonoid aglycones and preparation method thereof application

technical field

The invention belongs to the field of food, in particular to a guava leaf rich in soluble polyphenols and flavonoid aglycones and a preparation method and application.

Background technique

As a medicinal and edible substance, guava leaf has many years of use history, and has various effects such as anti-oxidation, inhibiting DNA damage, lowering blood sugar, anti-inflammatory, antibacterial, lowering blood pressure, and protecting the heart. Many studies have shown that the main bioactive functional components in guava leaves include polyphenols, which can eliminate the body damage caused by the damage of the antioxidant defense system caused by excess oxygen or nitrogen free radicals in the body. Since plant polyphenols mainly exist in plants in three forms (free state, conjugated state and bound state), and bound polyphenols are usually combined with polysaccharides and proteins on the plant cell wall in the form of chemical bonds, it is difficult to be extracted, resulting in low utilization of guava leaf polyphenolic active substances.

Therefore, it is necessary to promote the release of soluble polyphenols and flavonoid aglycones from guava leaves, so as to make full use of the active substances of guava leaf polyphenols.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a preparation method of guava leaves rich in soluble polyphenols and flavonoid aglycones.

Another object of the present invention is to provide a guava leaf product obtained by the above preparation method.

Another object of the present invention is to provide the application of the guava leaf product.

The object of the present invention is realized through the following technical scheme: a kind of preparation method of the guava leaf rich in soluble polyphenols and flavonoid aglycones, comprises the steps:

(1) the cleaned guava leaf is drained, dried, smashed, the smashed guava leaf is sieved, removes the guava leaf stem position, obtains the guava leaf position of substantially the same size;

(2) after the guava leaf part finally obtained in step (1) is mixed with water, adjust the pH value, add enzyme again, and carry out enzymolysis reaction;

(3) drying the system after the enzymatic hydrolysis reaction in step (2) to obtain a guava leaf product rich in soluble polyphenols and flavonoid aglycones.

The drying conditions described in step (1) are preferably drying at 50-80° C. to constant weight; more preferably, drying at 60° C. to constant weight.

The sieving described in the step (1) is preferably a 4-mesh sieve with an aperture.

The consumption of the water described in the step (2) is to disperse the guava leaves finally obtained in the step (1), so as to facilitate the enzymolysis reaction; preferably, it is equivalent to the quality of the guava leaves finally obtained in the step (1). 4 times.

The pH value described in step (2) is 4.5-6.0; preferably 5-5.5.

The temperature of the enzymatic hydrolysis reaction described in step (2) is 45-55°C; preferably 50°C.

The time of the enzymatic hydrolysis reaction described in step (2) is preferably 5-8 hours for each enzyme reaction; more preferably 6 hours for each enzyme reaction.

The enzyme described in step (2) is at least one of cellulase, hemicellulase, β-glucosidase and xylanase; preferably cellulase, hemicellulase and β-glucoside Combination use of enzymes.

The cellulase is preferably cellulase with an enzyme activity of 8000 U/g.

The hemicellulase is preferably a hemicellulase with an enzyme activity of 8000 U/g.

The β-glucosidase is preferably a β-glucosidase with an enzyme activity of 8000 U/g.

The xylanase is preferably a xylanase with an enzyme activity of 8000 U/g.

The mass dosage of the cellulase is preferably equivalent to 0.5% of the mass of the guava leaves.

The mass dosage of the hemicellulase is preferably equivalent to 0.5% of the mass of the guava leaves.

The mass dosage of the β-glucosidase is preferably equivalent to 0.5% of the mass of the guava leaves.

The mass dosage of the xylanase is preferably equivalent to 0.5% of the mass of the guava leaves.

The specific process of the enzymatic hydrolysis reaction is preferably shown in steps 1), 2) or 3), most preferably step 1):

1) First add cellulase for the first enzymatic hydrolysis to inactivate the cellulase; then add hemicellulase for the second enzymatic hydrolysis to inactivate the hemicellulase; finally add β-glucosidase for the third Second enzymatic hydrolysis to inactivate β-glucosidase;

2) firstly adding hemicellulase to carry out the first enzymatic hydrolysis to inactivate hemicellulase; then adding cellulase to carry out the second enzymatic hydrolysis to inactivate the cellulase; finally adding β-glucosidase to carry out the third enzymatic hydrolysis; Second enzymatic hydrolysis to inactivate β-glucosidase;

3) firstly adding β-glucosidase to carry out the first enzymatic hydrolysis to inactivate the β-glucosidase; then adding cellulase to carry out the second enzymatic hydrolysis to inactivate the cellulase; finally adding hemicellulase to carry out the first enzymatic hydrolysis; Three enzymatic hydrolysis to inactivate hemicellulase.

In steps 1), 2) and 3),

The reaction conditions of the first enzymatic hydrolysis, the second enzymatic hydrolysis and the third enzymatic hydrolysis are respectively preferably at 50°C for 6 hours;

The inactivation condition is preferably 80°C for 10min;

The mass dosage of described cellulase is preferably equivalent to 0.5% of the mass of the guava leaf part;

The mass dosage of the hemicellulase is preferably equivalent to 0.5% of the mass of the guava leaf part;

The mass dosage of the β-glucosidase is preferably equivalent to 0.5% of the mass of the guava leaves.

The drying temperature in step (3) is preferably 50-70°C; more preferably 60°C.

The drying time described in step (3) is preferably at least 12h; more preferably 16h.

A guava leaf product rich in soluble polyphenols and flavonoid aglycones is obtained by the above preparation method.

The guava leaf product rich in soluble polyphenols and flavonoid aglycones is used in the field of food and/or health care products; it can be directly eaten; it can also be further processed into various foods, such as rich in soluble polyphenols. Guava leaf tea beverage containing flavonoid aglycone, guava leaf biscuits rich in soluble polyphenols and flavonoid aglycone, nutritional meal bars, etc.

Compared with the prior art, the present invention has the following advantages and effects:

The preparation method provided by the invention is to release the insoluble bound polyphenols in the guava leaves that are not easy to extract through a variety of enzymatic hydrolysis, and convert them into easy-to-extract and soluble polyphenols; The ingredients are degraded into small molecules such as quercetin and kaempferol with higher absorption capacity and higher functional activity. The enzymatic hydrolysis reaction time is short, the conditions are mild, and the efficiency is high, which can be used for medicinal plant processing and synergy. The preparation method improves the antioxidant capacity of the guava leaf product, inhibits the DNA damage capacity, reduces blood sugar, cholesterol, and prevents cardiovascular and cerebrovascular diseases.

Description of drawings

Fig. 1 is a graph of the determination results of the total soluble polyphenol and insoluble polyphenol content of guava leaves in different embodiments.

Figure 2 is a graph of the measurement results of the total soluble flavonoids and insoluble flavonoids content of guava leaves in different embodiments.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1

(1) Preparation of guava leaf substrate: put the cleaned guava leaves into a 60 ℃ oven to dry for 16h, smash through a 4-mesh sieve, and the sieved guava leaves are the enzymatically hydrolyzed substrate; Water is added to the reaction-promoting matrix, and the amount of water (adjust pH=5.5 with citric acid) is 80% of the total weight;

(2) Enzymatic hydrolysis reaction: then mix cellulase (8000U/g, the same below) and the guava leaves finally obtained in step (1) and place them in a conical flask. Place in an oven at 80°C for 10min (cellulase inactivation), cool to room temperature; then add hemicellulase (8000U/g, the same below), mix well, enzymolysis for 6h in a water bath at 50°C, and place at 80°C Oven for 10min (inactivation of hemicellulase), cool to room temperature; continue to add β-glucosidase (8000U/g, the same below), mix well, enzymolysis for 6h in a 50°C water bath, and place in an 80°C oven 10min (deactivation β-glucosidase); wherein, the mass consumption of cellulase, hemicellulase, β-glucosidase is respectively equivalent to the sieved guava leaf (i.e. enzymatically catalyzed) obtained in step (1). 0.5% of the mass of the hydrolyzed matrix).

(3) Treatment of guava leaf products after enzymatic hydrolysis: The guava leaves after various enzymatic hydrolysis were dried in an oven at 60°C for 16 hours to obtain guava leaf products rich in soluble polyphenols and flavonoid aglycones.

Example 2

(1) Preparation of guava leaf substrate: basically the same as step (1) in Example 1, except that pH=5.5 was adjusted with citric acid.

(2) Multi-enzyme hydrolysis reaction: Next, mix the hemicellulase and the guava leaves finally obtained in step (1) and place them in a conical flask. After enzymatic treatment for 6 hours in a water bath at 50°C, place them in an oven at 80°C for 10 minutes. (deactivate hemicellulase), cool to room temperature; then add cellulase, mix well, enzymatically hydrolyze for 6 hours in a 50°C water bath, place in an oven at 80°C for 10 minutes (inactivate cellulase), and cool to room temperature; Continue to add β-glucosidase, mix evenly, enzymolysis for 6 hours in a 50°C water bath, and place in an oven at 80°C for 10 minutes (inactivating β-glucosidase); among them, hemicellulase, cellulase, β-glucosidase - The mass dosage of glucosidase is respectively equivalent to 0.5% of the mass of the sieved guava leaves (ie, the enzymatically hydrolyzed substrate) obtained in step (1).

(3) Treatment of guava leaf products after enzymatic hydrolysis: The guava leaves after various enzymatic hydrolysis were dried in an oven at 60°C for 16 hours to obtain guava leaf products rich in soluble polyphenols and flavonoid aglycones.

Example 3

(1) Preparation of guava leaf substrate: the same as step (1) in Example 2.

(2) Various enzymatic hydrolysis reactions: then mix xylanase (8000U/g) with the guava leaves finally obtained in step (1) and place them in a conical flask. Place in an oven at 80°C for 10min (inactivate xylanase), cool to room temperature; then add cellulase, mix well, under 50°C water bath, enzymolysis for 6h, place in an oven at 80°C for 10min (inactivate cellulase) ), cooled to room temperature; continued to add hemicellulase, mixed evenly, enzymatically hydrolyzed at 50°C for 6 hours, and placed in an oven at 80°C for 10 minutes (inactivating hemicellulase); among them, xylanase, fiber The mass dosages of vegetase and hemicellulase are respectively equivalent to 0.5% of the mass of the sieved guava leaves (ie, the enzymatically hydrolyzed substrate) obtained in step (1).

(3) Treatment of guava leaf products after enzymatic hydrolysis: The guava leaves after various enzymatic hydrolysis were dried in an oven at 60°C for 16 hours to obtain guava leaf products rich in soluble polyphenols and flavonoid aglycones.

Example 4

(1) Preparation of guava leaf substrate: the same as step (1) in Example 2.

(2) Various enzymatic hydrolysis reactions: Next, mix β-glucosidase and the guava leaves finally obtained in step (1) and place them in a conical flask. Oven for 10min (inactivation of β-glucosidase), cool to room temperature; then add cellulase, mix well, under 50°C water bath, enzymolysis for 6h, place in 80°C oven for 10min (inactivate cellulase), cool to room temperature; continue to add hemicellulase, mix evenly, enzymolysis for 6 hours in a 50°C water bath, and place in an oven at 80°C for 10 minutes (inactivation of hemicellulase); among them, β-glucosidase, cellulase The mass and dosage of hemicellulase are respectively equivalent to 0.5% of the mass of the sieved guava leaves (ie, the enzymatically hydrolyzed substrate) obtained in step (1).

(3) Treatment of guava leaf products after enzymatic hydrolysis: The guava leaves after various enzymatic hydrolysis were dried in an oven at 60°C for 16 hours to obtain guava leaf products rich in soluble polyphenols and flavonoid aglycones.

Effect Example

1. Detection method

The guava leaf products and untreated guava leaves prepared in Examples 1-4 were pulverized with a pulverizer and passed through a 40-mesh sieve for extraction and detection of the following components:

① Extraction of soluble polyphenols: Take 1.0 g of the guava leaf products prepared in Examples 1 to 4, respectively, in a 50 mL colorimetric tube, add 25 mL of 50% (v/v) methanol solution, and extract in a 45°C water bath for 1 hour. 0.45 μm filter paper was filtered, the filtrate was evaporated by a vacuum rotary evaporator at 37 ° C for 30 min to remove methanol to obtain a concentrated solution, 40 mL of distilled water was added to the concentrated solution, and then 10 mL of hexane was added to degreasing, and then extracted with 70 mL of ethyl acetate for 3 Next, the combined extracts were spin-dried in vacuo at 35°C to remove ethyl acetate. Finally, 5 mL of 50% (v/v) methanol was added to dissolve, which was the soluble polyphenol extract. Store at -20°C for polyphenol content analysis and HPLC quantitative analysis.

②Extraction of insoluble bound polyphenols: Add 40 mL of distilled water to the guava leaf residue remaining after the extraction of soluble polyphenols in step ① to remove the organic solvent, filter dry, dry at 60°C to constant weight, and record the weight of the residue. Add 40 mL of 4M NaOH solution, extract at room temperature for 4 hours, then adjust the pH to about 2 with concentrated hydrochloric acid (concentration of 37%), add 70 mL of ethyl acetate to extract 3 times, combine the extracts, spin dry at 35°C in vacuo to remove acetic acid Ethyl ester, and finally add 5 mL of 50% methanol to dissolve, which is the insoluble bound polyphenol extract. Store at -20°C for polyphenol content analysis and HPLC quantitative analysis.

③ Detection of polyphenol content: 100 μL of the above-extracted soluble polyphenol and insoluble bound polyphenol extract were respectively drawn and diluted to an appropriate concentration. Take 1mL of diluted sample solution or gallic acid standard solution (10-100μg/mL), add 0.5mL of Folin phenol reagent and mix, react for 3-8min, then add 1.5mL of 20% (w/v) Na ₂ CO ₃ solution, Add water to make the volume to 10mL, fully shake and mix, and let stand for 30min. Using blank reagent as control, measure the absorbance at 760nm.

④ Determination of flavonoids content: draw 100 μL of the soluble polyphenols and insoluble bound polyphenols extracts extracted above, and dilute to appropriate concentrations. Take 1 mL of diluted sample solution or rutin standard solution (10-100 μg/mL), add 0.3 mL of 5% (w/v) NaNO ₂ solution in turn, mix well, and let stand for 5 min. Add 0.3 mL of 10% (w/v) AlCl ₃ solution, mix well, and let stand for 6 min. Then add 2 mL of 4% (w/v) NaOH solution to mix well, add 70% (v/v) ethanol solution to make the volume to 10 mL, fully shake, and let stand for 10 min. With blank reagent as control, the absorbance value at 510nm was measured.

⑤ Detection of flavonoid aglycones (quercetin and kaempferol): respectively draw and filter the soluble polyphenols and insoluble bound polyphenols extracted above with 0.45μm filter paper, and filter the clear liquid through 0.22μm organic microfiltration. membrane, and the filtrate was subjected to HPLC analysis. The specific analysis conditions are: a high performance liquid chromatography system (Waters 2695) with an ultraviolet detector (Waters 2998), a detection wavelength of 350 nm, a column temperature of 30° C., and a C18 chromatographic column. The mobile phases used were: A-0.1% (v/v) formic acid aqueous solution, B-acetonitrile solution, the flow rate was 0.8 mL/min, and the injection volume was 10 μL. Detection conditions: gradient elution—0min, 85%A+15%B, 5min, 85%A+15%B, 10min, 80%A+20%B, 20min, 65%A+35%B, 30min, 50 %A+50%B, 31min, 20%A+80%B, 40min, 20%A+80%B, 45min, 85%A+15%B, 50min, 85%A+15%B (both by volume Compare). The analysis time was 50 min.

⑤ Antioxidant ability test:

a: DPPH free radical scavenging ability

Pipette 100 μL of the above-extracted soluble polyphenol and insoluble bound polyphenol extracts, respectively, and dilute to an appropriate concentration. Take 100 μL of diluted sample solution or vitamin C standard solution (5-30 μg/mL), add 400 μL of DPPH-methanol reagent, and let stand for 30 min at 30°C in the dark. Using water as a negative control and VC as a positive control, the absorbance at 510 nm was determined. The DPPH free radical scavenging ability of the sample is expressed as VC, that is, the mmol/L number of VC per g guava leaf sample is equivalent to.

b: ABTS ⁺ free radical scavenging ability

Pipette 100 μL of the above-extracted soluble polyphenol and insoluble bound polyphenol extracts, respectively, and dilute to an appropriate concentration. Take 50 μL of diluted sample solution or vitamin C standard solution (5-30 μg/mL), add 400 μL of ABTS ⁺ (7mM ABTS and 2.45mM K ₂ S ₂ O ₈ mixed in a volume ratio of 2:1, stand in the dark for 16h) reagent, 30°C, let stand in the dark for 30min. Using water as a negative control and VC as a positive control, the absorbance at 510 nm was determined. The ABTS ⁺ free radical scavenging ability of the sample is expressed as VC, that is, the mmol/L number of VC per g guava leaf sample is equivalent to.

⑥ Antioxidant ability test:

Take the above 2μL diluted soluble polyphenol and insoluble bound polyphenol extract solution (2mg/mL) and quercetin standard solution (2mg/mL) respectively, add 5μL pMD 18-T plasmid DNA (200ng/μL) , 10 μL of Fenton reagent (50 mM VC, 80 mM FeCl ₃ , and 30 mM H ₂ O ₂ ), mixed with a pipette, and left at 37° C. in the dark for 30 min. PBS buffer was used as blank control and quercetin was used as positive control, and then the mixture was added to 1% agarose gel for electrophoresis, and the DNA gel after electrophoresis was observed under ultraviolet conditions to calculate the ratio of helical DNA to total DNA. . The formula for calculating the DNA damage inhibition rate is as follows:

2. Test results

The results are shown in Figures 1 and 2. It was found that the soluble polyphenol content and the soluble flavonoid content of the guava leaf product after the enzyme treatment in the order of Examples 1 and 2 were significantly increased. The guava leaves treated with cellulase, hemicellulase and β-glucosidase had the highest soluble polyphenol content in the order of enzyme reaction in Example 1 (the guava leaves treated in Example 1 had the highest soluble polyphenol content relative to the untreated group). The content increased by 94.74%, the soluble flavonoids increased by 89.48%, and the content of insoluble bound polyphenols decreased significantly. This indicates that various enzyme treatments can promote the release of soluble polyphenols from guava leaves). However, the polyphenol release efficiency was the worst after adding xylanase, cellulase and hemicellulase enzymes sequentially in Example 3; Compared with the untreated group, the efficiency of obtaining soluble polyphenols from guava leaves is worse than that of Examples 1 and 2, but better than that of Example 3.

The content of flavonoid aglycones (quercetin and kaempferol) was detected by high performance liquid chromatography. After the sequential addition of multiple enzymes in Example 1 for co-hydrolysis, the contents of quercetin and kaempferol were the highest, which were 248.95mg/100g DM and 11.35mg/100g DM, respectively, which were increased by 1.97 times and 1.82 times, respectively, compared with the untreated group. . However, the total antioxidant activity of the guava leaf soluble polyphenol extract and the inhibitory effect on DNA damage were significantly improved after the various enzyme treatments of Examples 1 and 2. Cellulase, and 0.5% β-glucosidase) treated guava leaves with the highest biological activity. DPPH, ABTS ⁺ radical scavenging capacity is equivalent to 74.29 mmol VC/g DM, 77.41 mmol VC/g DM. The inhibition rate of DNA damage reached 81.23%. However, the guava leaves treated by the method of Example 3 had the lowest activity. The test data are shown in Table 1.

Table 1

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims (6)

1. A preparation method of guava leaves rich in soluble polyphenol and flavonoid aglycone is characterized by comprising the following steps:

(1) draining, drying, crushing and sieving the cleaned guava leaves, and removing the stems of the guava leaves to obtain the guava leaves with basically consistent sizes;

(2) mixing the guava leaf part finally obtained in the step (1) with water, adjusting the pH value, adding enzyme, and carrying out enzymolysis reaction;

(3) drying the system subjected to the enzymolysis reaction in the step (2) to obtain a guava leaf product rich in soluble polyphenol and flavonoid aglycone;

the specific process of the enzymolysis reaction is shown in steps 1), 2) or 3):

1) firstly, adding cellulase for carrying out first enzymolysis to inactivate the cellulase; adding hemicellulase for second enzymolysis to inactivate the hemicellulase; finally, adding beta-glucosidase to carry out third enzymolysis, and inactivating the beta-glucosidase;

2) adding hemicellulase for first enzymolysis to inactivate the hemicellulase; adding cellulase for second enzymolysis to inactivate the cellulase; finally, adding beta-glucosidase to carry out third enzymolysis, and inactivating the beta-glucosidase;

3) firstly, adding beta-glucosidase to carry out first enzymolysis, and inactivating the beta-glucosidase; adding cellulase for second enzymolysis to inactivate the cellulase; finally adding hemicellulase for carrying out third enzymolysis to inactivate the hemicellulase;

the cellulase has enzyme activity of 8000U/g;

the hemicellulase is the hemicellulase with the enzyme activity of 8000U/g;

the beta-glucosidase is beta-glucosidase with the enzyme activity of 8000U/g;

the mass consumption of the cellulase is equivalent to 0.5 percent of the mass of the guava leaf part;

the mass consumption of the hemicellulase is equivalent to 0.5 percent of the mass of the guava leaf part;

the mass usage amount of the beta-glucosidase is equivalent to 0.5 percent of the mass of the guava leaf part;

the pH value in the step (2) is 4.5-6.0;

the temperature of the enzymolysis reaction in the step (2) is 45-55 ℃;

and (3) the time of the enzymolysis reaction in the step (2) is counted by 5-8 hours of each enzyme reaction.

2. The method of claim 1, wherein the guava leaves are enriched in soluble polyphenols and flavonoid aglycones, and further comprising the steps of:

the drying condition in the step (1) is drying at 50-80 ℃ to constant weight;

the drying temperature in the step (3) is 50-70 ℃.

3. The method of claim 1, wherein the guava leaves are enriched in soluble polyphenols and flavonoid aglycones, and further comprising the steps of:

in the steps 1), 2) and 3),

the reaction conditions of the first enzymolysis, the second enzymolysis and the third enzymolysis are respectively 6 hours of reaction at 50 ℃;

the inactivation condition is that the treatment is carried out for 10min at 80 ℃.

4. A guava leaf product rich in soluble polyphenol and flavonoid aglycone is characterized in that: the preparation method of any one of claims 1 to 3.

5. Use of the guava leaf product enriched in soluble polyphenols and flavonoid aglycones as claimed in claim 4 for the preparation of a food product.

6. Use of the guava leaf product enriched in soluble polyphenols and flavonoid aglycones as claimed in claim 4 for the preparation of a health product.

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