Dendrobium nobile enzyme hydrolysate, and preparation method and application thereof

Abstract

The application provides a Dendrobium nobile Lindl. enzymatic hydrolysate and a preparation method and application thereof. The preparation method comprises the following steps: (1) crushing Dendrobium nobile Lindl., mixing and extracting with a solvent, filtering to obtain filter residues, mixing and extracting the filter residues with water, centrifugally collecting supernatant, mixing the supernatant with an alcohol solvent, redissolving the obtained precipitate with water, taking liquid to freeze-dry, and obtaining a Dendrobium nobile Lindl. extract; and (2) mixing the Dendrobium nobile Lindl. extract with an enzyme to perform enzymolysis, so as to obtain the Dendrobium nobile Lindl. enzymatic hydrolysate. The Dendrobium nobile Lindl. enzymatic hydrolysate provided by the application has the advantages of high antioxidant property, good hygroscopic effect and good absorbability.

Description

Technical Field

[0001] This invention belongs to the field of plant extract preparation, specifically relating to an enzymatic hydrolysis product of Dendrobium nobile, its preparation method and application, and particularly to an enzymatic hydrolysis product of Dendrobium nobile with good antioxidant properties, its preparation method and application. Background Technology

[0002] Dendrobium nobile, a perennial epiphytic herb belonging to the genus Dendrobium in the Orchidaceae family, contains various active ingredients such as Dendrobium polysaccharides and alkaloids. It possesses multiple medicinal properties, including nourishing yin and clearing heat, promoting body fluid production and benefiting the stomach, and moistening the lungs and relieving cough. Dendrobium polysaccharides are the main functional factors of Dendrobium, exhibiting antioxidant, moisturizing, and hypoglycemic activities. The bioactivity of Dendrobium nobile polysaccharides determines the broad development prospects of the Dendrobium polysaccharide industry. However, the large molecular weight of natural Dendrobium polysaccharides makes them difficult for organisms to absorb, limiting their application. Therefore, reducing the molecular weight of Dendrobium polysaccharides through appropriate methods to more effectively utilize their bioactivity is of great significance for their application.

[0003] CN111253495A discloses an extraction process for Dendrobium polysaccharides and Dendrobine from Dendrobium officinale, comprising the following steps: (1) washing, drying and pulverizing Dendrobium officinale; (2) supercritical CO2 extraction of Dendrobium officinale powder to obtain extract A; (3) extraction, vacuum concentration and drying of extract A to obtain Dendrobine; (4) centrifugation after soaking Dendrobium officinale residue in water, evaporation and concentration of supernatant to obtain extract B; (5) filtration of extract B into alcohol and retention of precipitate; (6) dissolving precipitate in distilled water and adding protease for enzymatic hydrolysis, adding trichloroacetic acid solution for static precipitation, filtering and collecting filtrate; (7) adding activated carbon to filtrate, static filtration and collection of filtrate, freeze-drying filtrate to obtain Dendrobium polysaccharides. This invention enables the stepwise extraction of Dendrobium polysaccharides and Dendrobine from Dendrobium officinale, with high extraction rates and high purity of the obtained Dendrobium polysaccharides and Dendrobine.

[0004] CN102786604A discloses a method for separating and extracting Dendrobium polysaccharides, dendrobine, and dendrobine from Dendrobium officinale in a single step. The method includes Dendrobium crushing, enzymatic hydrolysis, ultrasonic extraction, microfiltration, ultrafiltration and nanofiltration concentration, solid-liquid separation, vacuum low-temperature distillation, solvent extraction, solvent recovery, recrystallization, vacuum low-temperature drying, and supercritical carbon dioxide purification, finally yielding Dendrobium polysaccharides, dendrobine, and dendrobine. The products obtained by this invention have good color, good solubility, and high levels of active ingredients.

[0005] CN106282260A discloses an enzymatic-assisted extraction method for Dendrobium officinale polysaccharides. The method involves adding α-L-rhamnosidase enzyme preparation to a mixture of Dendrobium officinale and water for enzymatic hydrolysis, followed by water extraction and filtration. The supernatant is then concentrated by rotary evaporation and ethanol precipitation to obtain Dendrobium officinale polysaccharides. The technical advantages of this invention are: the addition of α-L-rhamnosidase enzyme preparation can increase the extraction rate of Dendrobium officinale polysaccharides, raising the extraction rate by up to 65.36% compared to the untreated rate; compared with other methods, the enzymatic-assisted extraction of polysaccharides is milder, easier to operate, more economical, and more effective, providing a foundation for the commercial application of Dendrobium officinale polysaccharides.

[0006] However, the above methods only focused on the extraction of Dendrobium polysaccharides and did not explore any aspects of improving their bioactivity. Therefore, how to provide a method that can effectively improve the performance of Dendrobium extract has become an urgent problem to be solved. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide an enzymatic hydrolysis product of Dendrobium nobile, its preparation method, and its application, particularly an enzymatic hydrolysis product of Dendrobium nobile with good antioxidant properties, its preparation method, and its application. The enzymatic hydrolysis product of Dendrobium nobile provided by the present invention has the advantages of high antioxidant properties, good hygroscopic effect, and good absorbability.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing enzymatic hydrolysis products of Dendrobium nobile, the preparation method comprising the following steps:

[0010] (1) Pulverize Dendrobium nobile, mix it with solvent for extraction, filter the residue and mix it with water for extraction, centrifuge and collect the supernatant and mix it with alcohol solvent, redissolve the obtained precipitate with water, freeze-dry the liquid to obtain Dendrobium nobile extract;

[0011] (2) The Dendrobium nobile extract was mixed with an enzyme and enzymatically hydrolyzed to obtain the Dendrobium nobile enzymatic hydrolysis product.

[0012] The above method can fully extract the active substances from Dendrobium nobile by using a specific extraction method, and can convert the macromolecules in the extract into active small molecules by using enzymatic hydrolysis, so that the product has excellent antioxidant and hygroscopic effects, and has the advantages of good absorbability.

[0013] Preferably, the solvent in step (1) includes ethyl acetate.

[0014] Preferably, the ratio of Dendrobium nobile to solvent is 1:(8-12)g / mL, such as 1:8g / mL, 1:9g / mL, 1:10g / mL, 1:11g / mL or 1:12g / mL, but not limited to the values ​​listed above. Other values ​​not listed above are also applicable.

[0015] Preferably, the extraction with solvent in step (1) is performed at least twice.

[0016] Preferably, the ratio of filter residue to water in step (1) is 1:(8-20)g / mL, such as 1:8g / mL, 1:9g / mL, 1:10g / mL, 1:11g / mL, 1:12g / mL, 1:13g / mL, 1:14g / mL, 1:15g / mL, 1:16g / mL, 1:17g / mL, 1:18g / mL, 1:19g / mL, or 1:20g / mL, but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0017] Preferably, the temperature for mixing and extraction with water is 80-100℃, such as 80℃, 85℃, 90℃, 95℃ or 100℃, but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0018] Preferably, the extraction with water in step (1) is performed at least twice.

[0019] Preferably, the alcohol solvent in step (1) includes anhydrous ethanol.

[0020] Preferably, the specific process of mixing with the alcohol solvent is to mix the supernatant with the alcohol solvent and let it stand for 8-16 hours, such as 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours or 16 hours, but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0021] Preferably, step (1) further includes concentrating the supernatant to obtain a concentrated solution before mixing with the alcohol solvent.

[0022] Preferably, the volume ratio of the concentrate to the alcohol solvent is 1:(3-5), such as 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0023] The specific extraction steps and parameters described above can effectively extract the active substances from Dendrobium nobile, improve the effect of subsequent enzymatic hydrolysis, and enhance the product's efficacy.

[0024] Preferably, the enzyme in step (2) includes any one or a combination of at least two of β-glucosidase, β-mannanase, α-galactosidase, α-amylase or cellulase, preferably a combination of α-galactosidase and α-amylase.

[0025] The selection of the aforementioned specific enzymes can effectively improve the enzymatic hydrolysis effect, fully converting the macromolecular active substances in the extract into small molecule active substances, effectively improving the absorbability of the product, and enhancing the product's antioxidant and hygroscopic effects.

[0026] Preferably, the ratio of Dendrobium nobile extract to α-galactosidase is 1:(0.5-3) mg / U, and the ratio of Dendrobium nobile extract to α-amylase is 1:(0.5-3) mg / KNU. The ratio of Dendrobium nobile extract to α-galactosidase can be 1:0.5 mg / U, 1:1 mg / U, 1:1.5 mg / U, 1:2 mg / U, 1:2.5 mg / U, or 1:3 mg / U, etc., and the ratio of Dendrobium nobile extract to α-amylase can be 1:0.5 mg / KNU, 1:1 mg / KNU, 1:1.5 mg / KNU, 1:2 mg / KNU, 1:2.5 mg / KNU, or 1:3 mg / KNU, etc., but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0027] Preferably, the enzymatic hydrolysis temperature in step (2) is 40-60℃, the pH is 5-8, and the hydrolysis time can be 8-15h. The temperature can be 40℃, 45℃, 50℃, 55℃ or 60℃, the pH can be 5, 5.5, 6, 6.5, 7, 7.5 or 8, and the time can be 8h, 9h, 10h, 11h, 12h, 13h, 14h or 15h, but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0028] Secondly, the present invention provides enzymatic hydrolysis products of Dendrobium nobile prepared by the preparation method described above.

[0029] Thirdly, the present invention also provides the application of the enzymatic hydrolysis products of Dendrobium nobile as described above in the preparation of cosmetics, health products or food.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention provides a method for preparing enzymatic hydrolysis products of Dendrobium nobile. By employing a specific extraction method, the active substances in Dendrobium nobile can be fully extracted, and the enzymatic hydrolysis can convert the macromolecules in the extract into active small molecules, resulting in a product with excellent antioxidant and hygroscopic effects, as well as good absorbability. Detailed Implementation

[0032] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0033] In the following examples, *Dendrobium nobile* was purchased from Chishui Zhilu Dendrobium nobile Ecological Park Development Co., Ltd.; α-galactosidase, α-amylase, cellulase, 1,1-diphenyl-2-trinitrobenzene, 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt, and quinoline dimethacrylate were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; β-glucosidase was purchased from Bailingwei Technology Co., Ltd.; β-mannanase was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; anhydrous sodium acetate was purchased from Shanghai Titan Technology Co., Ltd.; and anhydrous ethanol, ethyl acetate, and glacial acetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0034] Example 1

[0035] This embodiment provides an enzymatic hydrolysis product of Dendrobium nobile, and the specific steps are as follows:

[0036] Pulverize the dried Dendrobium nobile powder and pass it through a 40-mesh sieve. Weigh 50g of Dendrobium nobile powder, add 500mL of ethyl acetate, sonicate (300W, 40kHz) for 40min, and then filter. Sonicate the residue again with 500mL of ethyl acetate for 40min, and then filter. Let the residue stand overnight, and evaporate the ethyl acetate.

[0037] Add 750 mL of deionized water to the evaporated filter residue, boil for 1 hour, cool to 20°C, filter through a nylon mesh, and centrifuge the supernatant at 8000 rpm / min for 15 min. Collect the supernatant. Repeat the above hot water extraction operation on the precipitate, and combine the supernatants obtained from the two extractions. Rotary evaporate the supernatant to concentrate it to 250 mL.

[0038] Add 4 times the volume of anhydrous ethanol to the concentrated extract, stirring slowly at 500 rpm, and let stand at 4°C for 12 h. Collect the precipitate, wash twice with anhydrous ethanol, evaporate to dryness, redissolve in water, bring the volume to 250 mL, freeze-dry, and store to obtain Dendrobium nobile extract.

[0039] Take 200 mg of freeze-dried Dendrobium nobile polysaccharide, dissolve it in deionized water to prepare a 20 mg / mL aqueous solution, adjust the pH to 6.5, add 40 U / mL α-galactosidase and 20 KNU / mL α-amylase, react at 45℃ and 200 rpm for 12 hours, inactivate in a boiling water bath, centrifuge at 8500 rpm for 5 min, collect the supernatant and store it to obtain the Dendrobium nobile enzymatic hydrolysis product.

[0040] Example 2

[0041] This embodiment provides an enzymatic hydrolysis product of Dendrobium nobile, and the specific steps are as follows:

[0042] Pulverize the dried Dendrobium nobile powder and pass it through a 40-mesh sieve. Weigh 50g of Dendrobium nobile powder, add 400mL of ethyl acetate, sonicate (300W, 40kHz) for 40min, and then filter. Sonicate the residue again with 400mL of ethyl acetate for 40min, and then filter. Let the residue stand overnight, and evaporate the ethyl acetate.

[0043] Add 400 mL of deionized water to the evaporated filter residue, boil for 1 hour, cool to 20°C, filter through a nylon mesh, and centrifuge the supernatant at 8000 rpm / min for 15 min. Collect the supernatant. Repeat the above hot water extraction operation on the precipitate, and combine the supernatants obtained from the two extractions. Rotary evaporate the supernatant to concentrate it to 250 mL.

[0044] Add 3 times the volume of anhydrous ethanol to the concentrated extract, stirring slowly at 500 rpm, and let stand at 4°C for 8 hours. Collect the precipitate, wash twice with anhydrous ethanol, evaporate to dryness, redissolve in water, bring the volume to 250 mL, freeze-dry, and store to obtain Dendrobium nobile extract.

[0045] Take 200 mg of freeze-dried Dendrobium nobile polysaccharide, dissolve it in deionized water to prepare a 20 mg / mL aqueous solution, adjust the pH to 6.5, add 40 U / mL α-galactosidase and 20 KNU / mL α-amylase, react at 45℃ and 200 rpm for 12 hours, inactivate in a boiling water bath, centrifuge at 8500 rpm for 5 min, collect the supernatant and store it to obtain the Dendrobium nobile enzymatic hydrolysis product.

[0046] Example 3

[0047] This embodiment provides an enzymatic hydrolysis product of Dendrobium nobile, and the specific steps are as follows:

[0048] Pulverize the dried Dendrobium nobile powder and pass it through a 40-mesh sieve. Weigh 50g of Dendrobium nobile powder, add 600mL of ethyl acetate, sonicate (300W, 40kHz) for 40min, and then filter. Sonicate the residue again with 400mL of ethyl acetate for 40min, and then filter. Let the residue stand overnight, and evaporate the ethyl acetate.

[0049] Add 1000 mL of deionized water to the evaporated filter residue, boil for 1 hour, cool to 20°C, filter through a nylon mesh, and centrifuge the supernatant at 8000 rpm / min for 15 min. Collect the supernatant. Repeat the above hot water extraction operation on the precipitate, and combine the supernatants obtained from the two extractions. Rotary evaporate the supernatant to concentrate it to 250 mL.

[0050] Add 5 times the volume of anhydrous ethanol to the concentrated extract, stirring slowly at 500 rpm, and let stand at 4°C for 16 h. Collect the precipitate, wash twice with anhydrous ethanol, evaporate to dryness, redissolve in water, bring the volume to 250 mL, freeze-dry, and store to obtain Dendrobium nobile extract.

[0051] Take 200 mg of freeze-dried Dendrobium nobile polysaccharide, dissolve it in deionized water to prepare a 20 mg / mL aqueous solution, adjust the pH to 6.5, add 40 U / mL α-galactosidase and 20 KNU / mL α-amylase, react at 45℃ and 200 rpm for 12 hours, inactivate in a boiling water bath, centrifuge at 8500 rpm for 5 min, collect the supernatant and store it to obtain the Dendrobium nobile enzymatic hydrolysis product.

[0052] Example 4

[0053] This embodiment provides an enzymatic hydrolysis product of Dendrobium nobile. The specific steps are the same as in Example 1, except that the amount of α-amylase is adjusted to 10 KNU / mL.

[0054] Example 5

[0055] This embodiment provides an enzymatic hydrolysis product of Dendrobium nobile. The specific steps are the same as in Example 1, except that anhydrous ethanol is replaced with an equal amount of petroleum ether.

[0056] Example 6

[0057] This embodiment provides an enzymatic hydrolysis product of Dendrobium nobile. The specific steps are the same as in Example 1, except that α-galactosidase is replaced with an equal amount of α-amylase.

[0058] Example 7

[0059] This embodiment provides an enzymatic hydrolysis product of Dendrobium nobile. The specific steps are the same as in Example 1, except that α-amylase is replaced with an equal amount of α-galactosidase.

[0060] Example 8

[0061] This embodiment provides an enzymatic hydrolysis product of Dendrobium nobile. The specific steps are the same as in Example 1, except that α-galactosidase is replaced with an equal amount of cellulase.

[0062] Effect test:

[0063] 1. Determination of ABTS clearance rate

[0064] 7.7 mg of ABTS was dissolved in 2 mL of 2.45 mM potassium persulfate solution. After reacting for 16 h, an ABTS free radical stock solution was formed. Before use, the ABTS stock solution was diluted 40 times, and the absorbance at 734 nm was 0.7 ± 0.02. During the determination, 150 μL of ABTS working solution and 50 μL of test solution (5 mg / mL, solvent is water) were added to a 96-well plate, shaken to mix, and the absorbance of the reaction solution at 734 nm was measured after 10 min. Vc was used as a positive control. Three replicates were designed for the same determination. The scavenging rate was calculated by formula (1):

[0065] ABTS radical inhibition rate % = [1 - As / Ac)] × 100;

[0066] Where As represents the absorbance of the sample group; Ac represents the absorbance of the control group.

[0067] The products provided in Examples 1-8 were tested, and the results are as follows:

[0068] Example 1 78.5％ Example 2 68.2％ Example 3 51.9％ Example 4 71.7％ Example 5 40.2％ Example 6 45.6％ Example 7 53.3％ Example 8 72.4％

[0069] 2. Determination of DPPH scavenging rate

[0070] Weigh 8 mg DPPH and dissolve it in 100 mL of anhydrous ethanol (sonicated for 30 s). Dilute with Tris-HCl buffer 1:1 before use. For the assay, add 160 μL of DPPH working solution and 40 μL of the sample to be tested (5 mg / mL, water as solvent) to a 96-well plate, shake to mix, and measure the absorbance of the reaction solution at 517 nm after 30 min. Trolox was used as a positive control, and water as a control. Three replicates were designed for the same assay. The clearance rate was calculated using formula (2).

[0071] DPPH free radical scavenging rate % = [1 - (As-Ab) / Ac] × 100%;

[0072] Where As is the absorbance of the sample group; Ac is the absorbance of the control group; and Ab is the absorbance of the blank group.

[0073] The products provided in Examples 1-8 were tested, and the results are as follows:

[0074]

[0075]

[0076] 3. Measurement of moisture absorption effect

[0077] Accurately weigh 100 mg of the lyophilized sample into a weighing bottle. Place the weighing bottle open in a desiccator with a relative humidity of 43% and 81%, respectively. Take out and weigh the sample at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h. Perform 3 parallel sets for each sample.

[0078] Calculation of moisture absorption rate Ra: Ra (%) = (W t -W0) / W0×100%;

[0079] In the formula: W0 is the mass of the dried sample; W t The sample mass is t after moisture absorption time. The products provided in Examples 1-8 were tested, and the moisture absorption rate results after 8 hours are as follows:

[0080]

[0081] As can be seen from the above, the product provided by this invention has excellent antioxidant and hygroscopic effects. Comparative examples 1 and 4-5 show that by using specific extraction process reagents and enzyme parameters, this invention can fully extract the active substances from Dendrobium nobile, improve the effect of subsequent enzymatic hydrolysis, and enhance the antioxidant and hygroscopic effects of the product. Comparative examples 1 and 6-8 show that by selecting specific enzyme types, this invention further improves the enzymatic hydrolysis effect, enabling more complete conversion of macromolecular active substances in the extract into small molecule active substances, effectively improving the absorbability of the product and enhancing its antioxidant and hygroscopic effects.

[0082] The applicant declares that this invention illustrates the enzymatic hydrolysis product of Dendrobium nobile, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product, addition of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

[0083] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0084] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing enzymatic hydrolysate of Dendrobium nobile, characterized in that, The preparation method includes the following steps: (1) The Dendrobium nobile is pulverized, mixed with solvent for extraction, filtered, and the residue is mixed with water for extraction. The supernatant is collected by centrifugation and mixed with alcohol solvent. The precipitate is redissolved with water, and the liquid is freeze-dried to obtain Dendrobium nobile extract. The Dendrobium nobile extract is Dendrobium nobile polysaccharide. The temperature of the mixture of residue and water for extraction is 80-100℃. (2) The Dendrobium nobile extract is mixed with an enzyme and enzymatically hydrolyzed to obtain the Dendrobium nobile enzymatic hydrolysate; the enzyme is a combination of α-galactosidase and α-amylase; the ratio of Dendrobium nobile extract to α-galactosidase is 1:(0.5-3) mg / U, and the ratio of Dendrobium nobile extract to α-amylase is 1:(0.5-3) mg / KNU.

2. The preparation method according to claim 1, characterized in that, The solvent in step (1) includes ethyl acetate.

3. The preparation method according to claim 1, characterized in that, In step (1), the ratio of Dendrobium nobile to solvent is 1:(8-12) g / mL.

4. The preparation method according to claim 1, characterized in that, The ratio of filter residue to water in step (1) is 1:(8-20) g / mL.

5. The preparation method according to claim 1, characterized in that, The alcohol solvent in step (1) includes anhydrous ethanol.

6. The preparation method according to claim 1, characterized in that, The specific process of mixing with alcohol solvent in step (1) is to mix the supernatant with alcohol solvent and let it stand for 8-16 hours.

7. The preparation method according to claim 1, characterized in that, Step (1) further includes concentrating the supernatant to obtain a concentrated solution before mixing with the alcohol solvent.

8. The preparation method according to claim 7, characterized in that, The volume ratio of the concentrate to the alcohol solvent is 1:(3-5).

9. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis in step (2) is carried out at a temperature of 40-60℃, a pH of 5-8, and a time of 8-15 h.

10. An enzymatic hydrolysis product of Dendrobium nobile prepared by the preparation method according to any one of claims 1-9.

11. The application of the Dendrobium nobile enzymatic hydrolysis product according to claim 10 in the preparation of cosmetics, health products or food.

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